Multispecific antibody analogs comprising a common light chain, and methods of their preparation and use

ABSTRACT

Multispecific antibody analogs that co-engage at least two different antigens or epitopes (also referred to targets, used interchangeably throughout), said analogs comprising a common light chain, are provided, as well as methods for their production and use.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/973,830, filed Apr. 1, 2014, the disclosure of which is herebyincorporated by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 20, 2015, isnamed 2009186-0115 SL.txt and is 207,538 bytes in size.

FIELD OF THE INVENTION

The present invention relates, inter alia, to multispecific antibodyanalogs that comprise a common light chain, and methods of making andusing the same.

BACKGROUND OF THE INVENTION

All references cited herein, including patents, patent applications, andnon-patent publications referenced throughout are hereby expresslyincorporated by reference in their entireties for all purposes.

Antibodies and antibody-based molecules represent attractive candidatesas diagnostic tools and therapeutics. To date more than 30 therapeuticmonoclonal antibodies have been approved for and successfully applied indiverse indication areas including cancer, organ transplantation,autoimmune and inflammatory disorders, infectious disease, andcardiovascular disease.

However, the majority of these antibodies are monospecific antibodies,which recognize a single epitope and can be selected to either activateor repress the activity of a target molecule through this singleepitope. Many physiological responses, however, require crosslinking,“cross-talk” or co-engagement of or between two or more differentproteins or protein subunits to be triggered. An important example isthe activation of heteromeric, cell-surface receptor complexes. Forthese receptor complexes, activation is normally achieved through ligandinteraction with multiple domains on different proteins resulting inproximity-associated activation of one or both receptor components.

A desire to address and therapeutically exploit some of these morecomplex physiological processes, and disease states associatedtherewith, has stimulated significant effort towards generatingmultispecific antibodies that can co-engage multiple epitopes orantigens. One avenue that has received much attention is the engineeringof additional and novel antigen binding sites into antibody-based drugssuch that a single inventive multispecific antibody analog moleculeco-engages two or more different antigen targets (or distinct epitopeson one or more antigens). Such multispecific antibody analogs offer theability of consolidating the specificity of two epitopes into onemolecule. Within a therapeutic context, multispecific Abs may offeradditivity or synergy of efficacy depending upon the mechanism of actionand the nature of the target biology. However, several concerns havesurfaced related to manufacturing, and the ability to producemultispecific molecules that possess biophysical properties similar toapproved or clinically validated mAbs has plagued reduced the momentumof these molecules from entering the clinic. To address these concerns,several strategies have been proposed that may overcome issues relatedto aggregation and stability. However, these strategies do not offeroptions related to valency and may be limited in biological activity asa mechanism of cross-linking receptors. Alternative strategies thatcreate constructs able to engage two arms per target (tetravalent) mayoffer superior potency.

Tailoring valency for multispecific antibodies, such as bispecificantibodies (bsAbs) facilitates the identification of formats that canmatch with preferred targeting strategies. There exists a wide array ofconstructs that comprise different architectures which can have asignificant impact on engagement with selected targets. Severalbispecific constructs focus on the ability to engage each target withone arm of the antibody (monovalent), which may be essential fortargeting certain cell populations without activating the targetpathway. However, engagement with two arms for each target (bivalent)may the preferred method to bind to cell types in an avid state. Thisgenerally increases with affinity of the antibody to the target cell andfrequently increases the potency of antibodies. Bispecific antibodiesthat bind two targets bivalently are typically constructed astetravalent constructs by fusing variable regions together in a numberof formats. Single-chain antibodies (scFvs) have been applied in thismanner by fusing to IgGs to construct tetravalent bsAbs. While there aresuccessful reports of such constructs in which affinity and potency ofthe parental antibodies are retained, expression and biophysicalproperties are frequently impacted and create obstacles for drugdevelopment. Protein engineering techniques have been applied toovercome these obstacles, yet this is very labor intensive and notalways successful. While scFvs permit the ability to tailor valency withbispecifics, an alternative strategy to create tetravalent moleculesthat do suffer from production and stability issues would beadvantageous.

Fab fragments serve as an alternative source for adding specificity andtailoring valency. Fabs frequently exhibit production levels andstability similar to parental IgG and do not fall victim to theobstacles observed with scFvs. However, Fabs are larger moleculestypically expressed as two chains and are not connected with a linker aspresent with scFvs. As a result, generating bispecifics with Fabs can bechallenging depending on the format. Several reports in the literatureprovide opportunities to utilize the heterodimeric interface betweenVH:VL and CL:CH1, however, these molecules can be limited give theabsence of an Fc region that provides extended PK properties andeffector function attributed a glycosylated CH2 region.

The generation of multispecifics, such as bispecifics, containing Fabsand an Fc region has been achieved by several parties. To achieve highlevels of pure major product, two engineering obstacles must beaddressed: heavy chain and light chain promiscuity. IgG1 heavy chainsdimerize via the Fc region to form homodimers. To improve the pairing oftwo different heavy chains containing VH regions specific to twodifferent targets or epitopes, several groups have generated bispecificantibodies by modifying the CH3 domain to generate heterodimeric Fcconstructs. Employed strategies include substituting amino acids at theCH3 interface (Genentech, Amgen, Zymeworks), fusions with other IgGisotypes (EMD Serono), or substituting amino acids to modify Protein Abinding for purification of Fc-heterodimer bispecifics (see, e.g., U.S.Pat. No. 5,731,168; U.S. Pat. No. 7,643,228; U.S. Pat. No. 7,695,936;U.S. Pat. No. 8,216,805; U.S. Pat. No. 8,679,785; U.S. Pat. No.8,765,412; U.S. Pat. No. 7,951,917; U.S. Pat. No. 7,183,076; WO2013/0363702; WO 2014/012085). These constructs facilitate thegeneration of bispecifics in novel ways but do not address promiscuityof the light chains. This issue has been termed the “light chainproblem”. Several reports offer solutions by substituting amino acids atthe CL:CH1 and/or VH:VL interface. While early reports hold promise, amodular approach for screening bispecifics with germline diversityremains to be seen. As an alternative, several groups have identifiedtwo VH regions that bind to two different targets or epitopes yet sharethe same light chain (see, e.g., U.S. Pat. No. 5,731,168; U.S. Pat. No.7,643,228; U.S. Pat. No. 7,695,936; U.S. Pat. No. 8,216,805; U.S. Pat.No. 8,679,785; U.S. Pat. No. 8,765,412; U.S. Pat. No. 7,951,917; U.S.Pat. No. 7,183,076). This strategy reduces the number of lights chainsfrom two to one and reduces the number of chains for assembly from fourto three. However, the identification of VH regions that can share thesame light chain is not trivial. Issues related to compromise inaffinity and epitopic diversity are limiting factors to this approach.

Currently, there are several options for the identification of VHregions that share the same light chain. Fixed light chain repertoiresare available that offer the discovery of VH regions against differenttargets. However, this strategy requires the discovery against twotargets and may not be amenable to scenarios in which one existingantibody (HC and LC) is suited to be paired with another specificity.The ability to generate VH diversity surrounding any given light chain(rather than a pre-selected LC or an LC from a fixed light chainrepertoire, as described in, e.g., U.S. Pat. No. 7,919,257; U.S. Pat.No. 7,262,028; U.S. Pat. No. 7,927,834; U.S. Pat. No. 7,932,360; US2011/0250642; Merchant et al., Nature Biotechnol., 1998, 16: 677-681;Nissim et al., “Antibody fragments from a ‘single pot’ phage displaylibrary as immunochemical reagents,” EMBO J., 1994, 13: 692-698;Hoogenboom and Winter, J. Mol. Biol., 1992, 227, 381-388; Hoogenboom andWinter, “By-passing Immunisation,” J. Mol. Biol., 1992, 227: 381-388)would provide more opportunity to pair new specificities with antibodiesthat have already been validated (pre-clinical/clinical). In addition,the ability to bypass full-scale naïve discovery efforts or generatelarge diversities to minimize time and labor would provide an attractivealternative.

Others have reported yet further multispecific antibody analog formatsand methods for generating them (see, e.g., WO 2012/023053; WO 95/09917;WO 2013/177101; Miller et al., J. Immunol., Vol. 170, pages 4854-4861(2003); Coloma et al., Nature Biotechnology, Vol. 15, pages 159-163(1997); WO 2011/131746; WO 2012/123949).

There remains, therefore a need for multispecific antibody analogs that,for example, minimize some of the expression and production shortcomingsdescribed above and herein throughout. There also remains a need formultivalent and multispecific antibody analogs that allow for greaterflexibility in the number, orientation, and attachment points antigenbinding sites in the context of a multispecific antibody analog format.

SUMMARY OF THE INVENTION

Applicants have discovered, inter alia, multispecific antibody analogsand methods of making such, (referred to interchangeably throughout as“analogs” or “antibody analogs”), including bispecific, trispecific,tetraspecific, pentaspecific antibody analogs, and the like. Certainmultispecific antibody analogs in accordance with the invention mayadvantageously correspond to an IgG or IgG-like format, which formatcomprises a first polypeptide comprising a first heavy chain havingspecificity for a first antigen; second polypeptide comprising a secondheavy chain having specificity for a second antigen; and a common lightchain. Certain other multispecific antibody analogs in accordance withthe invention may advantageously comprise at least two copies of a firstpolypeptide and at least two copies of a second polypeptide, wherein thefirst polypeptide comprises a variable heavy domain (VH) withspecificity for one antigen (also referred interchangeably throughout as“target” or “targets”) of interest as well as a VH with specificity fora second antigen of interest, and wherein the second polypeptidecomprises a light chain variable domain that is compatible with both VHsand can for an antigen 1 binding region and an antigen 2 binding regionswhen associated with the first VH and the second VH, respectively.

Accordingly, in certain embodiments provided are methods of obtaining oridentifying one or more common light chains for use in preparing amultispecific antibody or multispecific antibody analog, the methodcomprising:

i) performing a first selection against a first antigen from a firstlibrary and obtaining one or more light chains from the output that hasspecificity for the first antigen;

ii) performing a second selection against a second antigen from a secondlibrary and obtaining heavy chains from the output that has specificityfor the second antigen;

iii) generating a restricted library comprising the one or more lightchains obtained in step i) and the heavy chains obtained in step ii);

iv) performing a third selection against the first antigen from therestricted library generated in step iii) and obtaining one or moreantibodies form the output of the third selection, wherein the one ormore antibodies comprise one or more light chains that each havespecificity for the first antigen and the second antigen;

thereby obtaining or identifying the one or more common light chains.

In certain embodiments, a) the first library comprises a naïve library;b) the second library comprises a naïve library; or c) the first librarycomprises a naïve library and the second library comprises a naïvelibrary. In further embodiments, such methods further comprise:

a) performing a subsequent selection against the first antigen from amaturation library;

b) performing a subsequent selection against the second antigen from amaturation library; or

c) performing a subsequent selection against the first antigen from amaturation library and performing a subsequent selection against thesecond antigen from a maturation library;

after performing:

a) step i;

b) step ii

c) step iii; and/or

d) step iv.

In certain other embodiments methods are provided for making amultispecific antibody analog comprising contacting the one or morecommon light chains obtained or identified according to any one theembodiments provided herein and throughout, wherein: a first polypeptidecomprising a heavy chain that has specificity for the first antigen; anda second polypeptide comprising a heavy chain that has specificity forthe second antigen. In certain of these embodiments, the one or morecommon light chains, the first polypeptide, and the second polypeptideare expressed by host cells. In certain of these embodiments, the one ormore common light chains, the first polypeptide, and the secondpolypeptide are expressed by the same host cell.

In certain other embodiments are provided a multispecific antibodyanalog comprising a common light chain obtained or identified byperforming a method as disclosed herein and throughout.

In certain other embodiments provided are methods of making amultispecific antibody analog comprising at least two first antigenbinding regions and at least two second antigen binding regions, saidfirst and second antigen binding regions having a common light chain,wherein first antigen binding regions have a different antigenspecificity than the second antigen binding regions, the methodcomprising:

i) obtaining at least one light chain from a first antigen bindingregion having specificity for the first antigen, wherein the firstantigen binding region comprises said at least one light chain and aheavy chain;

ii) obtaining heavy chains from the output of a selection performed froma naïve library against a second antigen;

iii) preparing a restricted library comprising heavy chains obtained instep ii) and the at least one light chain obtained in step i);

iv) performing a second selection against the second antigen from thelibrary prepared in step iii);

v) obtaining an multispecific antibody comprising the second antigenbinding region from the selection performed in step iv), wherein thesecond antigen binding region comprises the at least one light chainobtained in step i);

vi) incorporating the first antigen binding region and the secondantigen binding region into a multispecific antibody format, wherein theformat comprises:

an IgG moiety comprising either:

a) the first antigen binding region; or

b) the second antigen binding region; and

two Fab moieties, wherein each Fab moiety comprises either:

a) the second antigen binding region; or

b) the first antigen binding region;

wherein the N-terminus of the heavy chain of one Fab moiety is linked tothe C-terminus of the Fc region of one heavy chain of the IgG moiety viaa linker moiety, and the N-terminus of the heavy chain of the other Fabmoiety is linked to the C-terminus of the Fc region of the other heavychain of the IgG moiety via a linker moiety; thereby generating themultispecific antibody analog.

In certain embodiments, optionally in combination with any of thepreceding or following embodiments, each linker moiety independentlycomprises a peptide from 1 to 75 amino acids in length, inclusive. Incertain embodiments, one or more of the linker moieties independentlycomprises at least one of the 20 naturally occurring amino acids. Inother embodiments, optionally in combination with any of the precedingor following embodiments, one or more of the linker moietiesindependently comprises at least one non-natural amino acid incorporatedby chemical synthesis, post-translational chemical modification or by invivo incorporation by recombinant expression in a host cell. In certainother embodiments, optionally in combination with any of the precedingor following embodiments, one or more of the linker moietiesindependently comprises one or more amino acids selected from the groupconsisting of serine, glycine, alanine, proline, asparagine, glutamine,glutamate, aspartate, and lysine. In other embodiments, optionally incombination with any of the preceding or following embodiments, one ormore of the linker moieties independently comprises a majority of aminoacids that are sterically unhindered. In other embodiments, optionallyin combination with any of the preceding or following embodiments, oneor more of the linker moieties independently comprises one or more ofthe following: an acidic linker, a basic linker, and a structural motif.In other embodiments, optionally in combination with any of thepreceding or following embodiments, one or more of the linker moietiesindependently comprises: polyglycine, polyalanine, poly(Gly-Ala), orpoly(Gly-Ser). In other embodiments, optionally in combination with anyof the preceding or following embodiments, one or more of the linkermoieties independently comprises: a polyglycine selected from the groupconsisting of: (Gly)₃ (SEQ ID NO: 1), (Gly)₄ (SEQ ID NO: 2), and (Gly)₅(SEQ ID NO: 3). In other embodiments, optionally in combination with anyof the preceding or following embodiments, one or more of the linkermoieties independently comprises (Gly)₃Lys(Gly)₄ (SEQ ID NO: 4);(Gly)₃AsnGlySer(Gly)₂ (SEQ ID NO: 5); (Gly)₃Cys(Gly)₄ (SEQ ID NO: 6);and GlyProAsnGlyGly (SEQ ID NO: 7). In other embodiments, optionally incombination with any of the preceding or following embodiments, one ormore of the linker moieties independently comprises a combination of Glyand Ala. In other embodiments, optionally in combination with any of thepreceding or following embodiments, one or more of the linker moietiesindependently comprises a combination of Gly and Ser. In otherembodiments, optionally in combination with any of the preceding orfollowing embodiments, one or more of the linker moieties independentlycomprises a combination of:

Gly and Glu; or Gly and Asp. In other embodiments, optionally incombination with any of the preceding or following embodiments, one ormore of the linker moieties independently comprises a combination of Glyand Lys.

In other embodiments, optionally in combination with any of thepreceding or following embodiments, one or more of the linker moietiesindependently comprises a sequence selected from group consisting of:[Gly-Ser]n (SEQ ID NO: 8); [Gly-Gly-Ser]n (SEQ ID NO: 9);[Gly-Gly-Gly-Ser]n (SEQ ID NO: 10); [Gly-Gly-Gly-Gly-Ser]n (SEQ ID NO:11); [Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly]n (SEQ ID NO: 12);[Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly]n (SEQ ID NO:13); [Gly-Gly-Gly-Gly-SerGly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly]n (SEQ ID NO:14);[Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly]n(SEQ ID NO: 15);[Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly]n(SEQ ID NO: 16);[Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly]n(SEQ ID NO: 17); and combinations thereof; where n is an integerselected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, and 75.

In other embodiments, optionally in combination with any of thepreceding or following embodiments, one or more of the linker moietiesindependently comprises a sequence selected from the group consistingof: [Gly-Glu]n (SEQ ID NO: 18); [Gly-Gly-Glu]n (SEQ ID NO: 19);[Gly-Gly-Gly-Glu]n (SEQ ID NO: 20); [Gly-Gly-Gly-Gly-Glu]n (SEQ ID NO:21); [Gly-Asp]n (SEQ ID NO: 22); [Gly-Gly-Asp]n (SEQ ID NO: 23);[Gly-Gly-Gly-Asp]n (SEQ ID NO: 24); [Gly-Gly-Gly-Gly-Asp]n (SEQ ID NO:25); and combinations thereof; where n is an integer selected from thegroup consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, and 75.

In other embodiments, optionally in combination with any of thepreceding or following embodiments, at least one of the first and secondantigen binding regions comprises at least one humanized variable heavydomain or at least one humanized variable light domain. In otherembodiments, optionally in combination with any of the preceding orfollowing embodiments, at least one of the first and second antigenbinding regions comprises at least one complimentary determining regionCDR that is derived from a non-human multispecific antibody ormultispecific antibody fragment.

In other embodiments, optionally in combination with any of thepreceding or following embodiments, at least one of the first and secondantigen binding regions binds an epitope from a tumor associatedantigen, a hormone receptor, a cytokine receptor, chemokine receptor, agrowth factor receptor, an immune activating receptor, a hormone, acytokine, a chemokine, a growth factor, a G protein-coupled receptor, ora transmembrane receptor. In other embodiments, optionally incombination with any of the preceding or following embodiments, leastone of the first and second antigen binding regions binds a targetassociated with an autoimmune disorder, an inflammatory disorder, anoncological disorder, neuromuscular disorder, a neurodegenerativedisorder, a metabolic disorder, or an infectious disease.

In other embodiments, optionally in combination with any of thepreceding or following embodiments the multispecific antibody analogbinds at least two different targets. In other embodiments, optionallyin combination with any of the preceding or following embodiments, themultispecific analog binds at least three different targets. In otherembodiments, optionally in combination with any of the preceding orfollowing embodiments, the multispecific antibody analog binds at leastfour different targets. In other embodiments, optionally in combinationwith any of the preceding or following embodiments the multispecificantibody analog binds at least one target monovalently. In otherembodiments, optionally in combination with any of the preceding orfollowing embodiments, the multispecific antibody analog binds at leasttwo targets monovalently. In other embodiments, optionally incombination with any of the preceding or following embodiments, themultispecific antibody analog binds at least three targets monovalently.In other embodiments, optionally in combination with any of thepreceding or following embodiments, the multispecific antibody analogbinds at least four targets monovalently.

In other embodiments, optionally in combination with any of thepreceding or following embodiments, at least one of the antigen bindingregions comprises or is derived from a non-human species. In otherembodiments, optionally in combination with any of the preceding orfollowing embodiments, at least one of the antigen binding sitescomprises a humanized variable domain or a humanized CDR.

In other embodiments, optionally in combination with any of thepreceding or following embodiments, at least one VH comprises a VH CDR1,a VH CDR2, and a VH CDR3 each independently selected from the following:a VH CDR1 amino acid sequence selected from the group consisting of:GSVSSGSYYWS (SEQ ID NO: 26); GSISSGGYYWS (SEQ ID NO: 27); GSINSSSYYWQ(SEQ ID NO: 28); FTLSGDWIH (SEQ ID NO: 29); FNIKDTYIH (SEQ ID NO: 30);FSLTNYGVH (SEQ ID NO: 31); GSISSGGDYWQ (SEQ ID NO: 32); a VH CDR2 aminoacid sequence selected from the group consisting of: YIYYSGSTNYNPSLKS(SEQ ID NO: 33); IIYYSGWTNYNPSLKS (SEQ ID NO: 34); EIAYSGSTYYNPSLKS (SEQID NO: 35); EISAAGGYTDYADSVKG (SEQ ID NO: 36); RIYPTNGYTRYADSVKG (SEQ IDNO: 37); VIWSGGNTDYNTPFTSR (SEQ ID NO: 38); and a VH CDR3 selected fromthe group consisting of: ARTNLYSTPFDI (SEQ ID NO: 39);ARGVGPDFWSGYSYSSYFDL (SEQ ID NO: 40); ARGQQWAAFDI (SEQ ID NO: 41);ARESRVSFEAAMDY (SEQ ID NO: 42); SRWGGDGFYAMDY (SEQ ID NO: 43);RALTYYDYEFAYW (SEQ ID NO: 44).

In other embodiments, optionally in combination with any of thepreceding or following embodiments, at least one VL comprises a VL CDR1,a VL CDR2, and a VL CDR3 each independently selected from the following:a VL CDR1 amino acid sequence selected from the group consisting of:RASQDISSWLA (SEQ ID NO: 45); RASQAISSWLA (SEQ ID NO: 46); RASQNIATDVA(SEQ ID NO: 47); RASQDVNTAVA (SEQ ID NO: 48); RASQSIGTNIH (SEQ ID NO:49); a VL CDR2 amino acid sequence selected from the group consistingof: AASSLQS (SEQ ID NO: 50); DASSLES (SEQ ID NO: 51); AASSLQS (SEQ IDNO: 52); SASFLYS (SEQ ID NO: 53); YASESIS (SEQ ID NO: 54); and a VL CDR3amino acid sequence selected from the group consisting of: QQEHDFPWT(SEQ ID NO: 55); HQYQSYSWT (SEQ ID NO: 56); QQEHDFPWT (SEQ ID NO: 57);QQSEPEPYT (SEQ ID NO: 58); QQHYTTPPT (SEQ ID NO: 59); QQNNNWPTT (SEQ IDNO: 60).

In other embodiments, optionally in combination with any of thepreceding or following embodiments, the multispecific antibody analogcomprises at least one heavy chain framework region that corresponds toor is derived from VH1-46, VH3-23, VH4-39, or VH4-61, and wherein atleast one light chain framework region that corresponds to or is derivedfrom VK1-05, VK1-12, or VK3-11.

In other embodiments, optionally in combination with any of thepreceding or following embodiments, the multispecific antibody analogcomprises a VH region that comprises an amino acid sequence selectedfrom the group consisting of:

(SEQ ID NO: 61) VQLQESGPGLVKPSETLSLTCTVSGGSVSSGSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR TNLYSTPFDIWGQGTMVTVSS;(SEQ ID NO: 62) QVQLQESGPGLVKPSETLSLTCTVSGGSISSGGYYWSWIRQPPGKGLEWIGIIYYSGWTNYNPSLKSRVTISVDASRNQFSLKLSSVTAADTAVYYCARGVGPDFWSGYSYSSYFDLWGRGTLVTVSS; (SEQ ID NO: 63)QLQLQESGPGLVKPSETLSLTCTVSGGSINSSSYYWQWIRQPPGKGLEWIGEIAYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCA RGQQWAAFDIWGQGTMVTVSS;(SEQ ID NO: 64) EVQLVESGGGLVQPGGSLRLSCAASGFTLSGDWIHWVRQAPGKGLEWVGEISAAGGYTDYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARESRVSFEAAMDYWGQGTLVTVSS; (SEQ ID NO: 65)EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS; (SEQ ID NO: 66)QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSDDTAIYYCARA LTYYDYEFAYWGQGTLVTVSS;and (SEQ ID NO: 67) QLQLQESGPGLVKPSETLSLTCTVSGGSISSGGDYWQWIRQPPGKGLEWIGEIAYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCA RGQQWAAFDIWGQGTMVTVSS.

In other embodiments, optionally in combination with any of thepreceding or following embodiments, the multispecific antibody analogcomprises a VL region that comprises an amino acid sequence selectedfrom the group consisting of:

(SEQ ID NO: 68) DIQLTQSPSSVSASVGDRVTITCRASQDISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQEHDFPWTF GGGTKVEIK;(SEQ ID NO: 69) DIQLTQSPSTLSASVGDRVTITCRASQAISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCHQYQSYSWTF GGGTKVEIK;(SEQ ID NO: 70) DIQLTQSPSSVSASVGDRVTITCRASQDISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQEHDFPWTF GGGTKVEIK;(SEQ ID NO: 71) DIQMTQSPSSLSASVGDRVTITCRASQNIATDVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSEPEPYTF GQGTKVEIK;(SEQ ID NO: 72) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTF GQGTKVEIK; and(SEQ ID NO: 73) DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTHGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTF GAGTKLELK.

In other embodiments, optionally in combination with any of thepreceding or following embodiments, the multispecific antibody analogcomprises a polypeptide comprising, from N-terminus to C-terminus, afirst VH region, a CH1, a hinge region, a CH2 region, a CH3 region, asecond VH region, and a CH1 region, the amino acid sequence of whichcomprises an amino acid sequence selected from the group consisting of:

(SEQ ID NO: 74) QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARTNLYSTPFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSQLQLQESGPGLVKPSETLSLTCTVSGGSINSSSYYWQWIRQPPGKGLEWIGEIAYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGQQWAAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC; (SEQ ID NO: 75)QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARTNLYSTPFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGGSISSGGYYWSWIRQPPGKGLEWIGIIYYSGWTNYNPSLKSRVTISVDASRNQFSLKLSSVTAADTAVYYCARGVGPDFWSGYSYSSYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSC;(SEQ ID NO: 76) QVQLQESGPGLVKPSETLSLTCTVSGGSISSGGYYWSWIRQPPGKGLEWIGIIYYSGWTNYNPSLKSRVTISVDASRNQFSLKLSSVTAADTAVYYCARGVGPDFWSGYSYSSYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGGSVSSGSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARTNLYSTPFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSC;(SEQ ID NO: 77) QLQLQESGPGLVKPSETLSLTCTVSGGSINSSSYYWQWIRQPPGKGLEWIGEIAYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGQQWAAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGGSVSSGSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARTNLYSTPFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS C.

In other embodiments, optionally in combination with any of thepreceding or following embodiments, the multispecific antibody analogcomprises four copies of a polypeptide comprising, from N-terminus toC-terminus, a VL region, and a CK region, and wherein said polypeptideheterodimerizes with compatible VH regions of the multispecific antibodyanalog, the amino acid sequence of which comprises an amino acidsequence selected from the group consisting of:

(SEQ ID NO: 78) DIQLTQSPSSVSASVGDRVTITCRASQDISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQEHDFPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC;(SEQ ID NO: 79) DIQLTQSPSTLSASVGDRVTITCRASQAISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCHQYQSYSWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC;(SEQ ID NO: 80) DIQMTQSPSSLSASVGDRVTITCRASQNIATDVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSEPEPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC;(SEQ ID NO: 81) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC;and (SEQ ID NO: 82) DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTHGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC.

In other embodiments, optionally in combination with any of thepreceding or following embodiments, the multispecific antibody analoghas binding specificity for an oncology target. In other embodiments,optionally in combination with any of the preceding or followingembodiments, the multispecific antibody analog has binding specificityfor one or more targets selected from the group consisting of: EGFR,HER2, and HER3. In other embodiments, optionally in combination with anyof the preceding or following embodiments, the multispecific antibodyanalog has binding specificity for EGFR and HER2. In other embodiments,optionally in combination with any of the preceding or followingembodiments, the polypeptide multispecific antibody analog has bindingspecificity for EGFR and HER3. In other embodiments, optionally incombination with any of the preceding or following embodiments, themultispecific antibody analog has binding specificity for EGFR, HER2,and HER3.

In other embodiments, optionally in combination with any of thepreceding or following embodiments, the multispecific antibody analog isselected from the group consisting of the multispecific antibody analogsdescribed in the Examples.

In other embodiments, optionally in combination with any of thepreceding or following embodiments, the multispecific antibody analog isexpressed by a prokaryotic host cell or a eukaryotic host cell. In otherembodiments, optionally in combination with any of the preceding orfollowing embodiments, the multispecific antibody analog is expressed bya eukaryotic host cell. In other embodiments, optionally in combinationwith any of the preceding or following embodiments, the multispecificantibody analog is expressed by a eukaryotic host cell selected from thegroup consisting of: yeast cells; Saccharomyces cerevisiae cells; Pichiacells; mammalian cells; Chinese hamster ovary (CHO) cells; humanembryonic kidney (HEK) cells; insect cells; Sf9 cells; and Sf21 cells.

In other embodiments, optionally in combination with any of thepreceding or following embodiments, provided are multispecific antibodyanalogs prepared by performing a method according to any of thepreceding or following embodiments.

As the artisan will understand, any and all of the embodiments disclosedabove and throughout may be practiced in any combination and,accordingly, all such combinations are contemplated, and are herebydisclosed and encompassed within the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a schematic representation of an exemplary naïve libraryantigen selection scheme for identifying antibodies with specificity forAntigen.

FIG. 2 provides the dissociation constants (K_(D)) measured using a MesoScale Discovery (MSD)-based assay (see, e.g., Estep et al., MAbs, Vol.5(2), pp. 270-278 (2013)) for five IgG antibodies that were isolated byperforming a selection, as illustrated in FIG. 1, for EGFR binders usinga naïve library. M=molar.

FIG. 3 illustrates and exemplary selection scheme for identifying one ormore light chains that can pair with a first heavy chain havingspecificity for an Antigen 1 as well as a second heavy chain havingspecificity for an Antigen 2 (i.e., a “common light chain”). A firstselection using a naïve library is performed against an Antigen 1 (e.g.,EGFR), and one or more light chains from one or more IgGs is isolatedfrom the first selection. A second selection using a naïve library isperformed against Antigen 2 (e.g., HER2), and heavy chains are obtained(“rescued”) from the output of binders having specificity for Antigen 2isolated from the second selection. The heavy chains (or a subset ofsuch heavy chains) are added with the one or more light chains from thefirst selection, generating a restricted library that is enriched forheavy chains having selectivity for Antigen 2 and light chains havingspecificity for Antigen 1. The total diversity of this restrictedlibrary is typically about: 10⁹, or less, 10⁸, or less, or 10⁷ or less;and in each case, the vast majority of the diversity is comprised of theheavy chains. A subsequent selection is performed against Antigen 2using this restricted library, and the binders having specificity forAntigen 2 are obtained.

FIG. 4 illustrates an exemplary selection scheme in which a common lightchain (common LC) was identified that is compatible with heavy chainsidentified in naïve selections against EGFR and HER2, respectively. Theaffinities of the EGFR-binding IgGs from which the 5 denoted lightchains were obtained were as illustrated in FIG. 2. The affinity of theprogeny obtained by performing the subsequent selections against HER2using the restricted library is provided (KD=190 pM). Results ofexperiments to determine whether such progeny could effectively bind tonative HER2 antigen expressed on BT474 cells is provided, and was foundto be significantly greater than that obtained by using Herceptin(88,834 MFI vs., 25,610 MFI, respectively).

FIG. 5 illustrates alternative approaches and formats that may beemployed in which common light chains identified in accordance with thedisclosed methods may be applied in order to generate multispecificanalogs as disclosed and claimed. Fab=antibody-binding fragment.

FIG. 6 depicts the affinities of anti-EGFR and HER2 binders eachcomprising a common light chain and each identified in accordance withmethods illustrated in FIGS. 1 through 3, as well as the IgG-Fab 2+2format was generated therewith, as disclosed in the Examples.

FIG. 7 provides a comparison between the binding affinities of each EGFRbinding region and HER2 binding region in the context of the individualIgGs, as provided in FIG. 6, and the binding affinities of each antigenbinding region in the context of the illustrated multispecific antibodyanalogs (bottom two IgG-Fab constructs), as disclosed, e.g., in theExamples.

FIG. 8 provides size exclusion chromatographic (SEC) profiles obtainedof the individual EGFR- and HER2-binding IgGs depicted in FIGS. 5 and 6in comparison with the SEC profiles obtained for the multispecificantibody analogs that are depicted in FIG. 6.

FIG. 9 provides an assessment of the melting temperature (T_(M))obtained for each of the individual EGFR- and HER2-IgGs depicted inFIGS. 5 and 6 in and the multispecific antibody analogs that aredepicted in FIG. 6.

FIG. 10 illustrates an exemplary selection scheme in which a commonlight chain (common LC) was identified that is compatible with heavychains identified in naïve selections against EGFR and HER3,respectively. The affinities of the EGFR-binding IgGs from which the 5denoted light chains were obtained were as illustrated in FIG. 2. Theaffinity of the progeny obtained by performing the subsequent selectionsagainst HER3 using the restricted library is provided (KD=39 pM).Results of experiments to determine whether such progeny couldeffectively bind to native HER3 antigen expressed on MDA-mb-453 cells isprovided, and was found to be comparable to that obtained by usingDuligotumab (Genentech) (4,353 MFI vs., 4,309 MFI, respectively).

FIG. 11 depicts the affinity of the Anti-EGFR from which the commonlight chain was isolated in order to identify an anti-HER3-binding IgGin accordance with the methods disclosed herein.

FIG. 12 provides a comparison between the binding affinities of eachEGFR binding region and HER3 binding region, identified in accordancewith the disclosed methods (see, e.g., FIG. 3 and the Examples) in thecontext of the individual IgGs, and the binding affinities of eachantigen binding region in the context of the illustrated multispecificantibody analogs (bottom two IgG-Fab constructs), as disclosed, e.g., inthe Examples.

FIG. 13 provides size exclusion chromatographic (SEC) profiles obtainedof the individual EGFR- and HER3-binding IgGs depicted in FIGS. 10through 12, in comparison with the SEC profiles obtained for themultispecific antibody analogs that are depicted in FIG. 12.

FIG. 14 provides an assessment of the melting temperature (T_(M))obtained for each of the individual EGFR- and HER2-IgGs depicted inFIGS. 10 through 12 in and the multispecific antibody analogs that aredepicted in FIG. 12.

FIG. 15 depicts the identification of a common light chain (common LC)that is able to pair with heavy chains identified from separate naïvelibrary selections against EGFR, HER2, and HER3, respectively, inaccordance with the methods disclosed throughout (e.g., in theExamples), and as illustrated in FIGS. 2, 3, 10, and 11.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides, inter alia, multispecific antibodyanalogs (referred to interchangeably throughout as “multispecificanalogs”, “analogs” or “antibody analogs”), including bispecific,trispecific, tetraspecific, pentaspecific antibody analogs, and thelike, which advantageously comprise at least two copies of a firstpolypeptide and at least two copies of a second polypeptide, whereinsuch multispecific analogs have specificity for more than one antigen(also referred interchangeably throughout as a “target” or in the pluralsense “targets”).

As would be understood by those of ordinary skill in the art, the term“antibody” is used herein in the broadest sense and specificallyencompasses at least monoclonal antibodies, polyclonal antibodies,multi-specific antibodies (e.g., bispecific antibodies), multispecificantibody analogs, chimeric antibodies, humanized antibodies, humanantibodies, antibody fragments, and derivatives thereof. An antibody isa protein comprising one or more polypeptides substantially or partiallyencoded by immunoglobulin genes or fragments of immunoglobulin genes.The recognized immunoglobulin genes include the kappa, lambda, alpha,gamma, delta, epsilon and mu constant region genes, as well as myriadimmunoglobulin variable region genes. An “antibody” also refers to animmunoglobulin molecule, a fragment of an immunoglobulin molecule, or aderivative thereof, which has the ability to specifically bind to anantigen, which may be, for example: a protein; a polypeptide; peptide; ahormone; a cytokine; a chemokine; a growth factor; a neurotransmitter; acarbohydrate-containing biological molecule; a lipid or fattyacid-containing biological molecule; or other biological molecule; viaan epitope present on such antigen. An “antibody analog” or “antibodyanalogs” refers to variants, of an antibody or antibody format. Suchantibody analogs may comprise variants or variations with regard to theformat, structure, or amino acid composition, or antibodies, asdescribes herein and throughout.

Multispecific antibody analogs that may be generated or used inaccordance with the invention may correspond to a variety of differentformats. In certain embodiments, multispecific analogs may compriseantibodies that comprise or otherwise correspond to an Ig-like format,such as an IgG, IgA, IgD, or IgM format. In embodiments in which themultispecific analogs comprise or otherwise correspond to, for example,an IgG format, such antibody analogs comprise: a first polypeptidecomprising a first heavy chain having specificity for a first antigen(also referred interchangeably throughout as an “antigen 1”); a secondpolypeptide comprising a second heavy chain having specificity for asecond antigen (also referred interchangeably throughout as an “antigen2”); and a third polypeptide comprising a common light chain. In certainof these embodiments, the first polypeptide comprises a first variableheavy domain (VH) with specificity for the first antigen of interest;the second polypeptide comprises a second variable heavy domain (VH)with specificity for the second antigen of interest; and the thirdpolypeptide comprises a light chain variable domain that is compatiblewith both VHs and can form a first antigen binding region and a secondantigen binding region when associated with the first VH and the secondVH, respectively.

Also provided herein are methods by which one or more common lightchains may be obtained or identified for use in preparing multispecificantibody analogs in accordance with the invention, which comprise: i)performing a first selection against a first antigen from a firstlibrary and obtaining one or more light chains from the output that hasspecificity for the first antigen; ii) performing a second selectionagainst a second antigen from a second library and obtaining heavychains from the output that has specificity for the second antigen; iii)generating a restricted library comprising the one or more light chainsobtained in step i) and the heavy chains obtained in step ii); iv)performing a third selection against the first antigen from therestricted library generated in step iii) and obtaining one or moreantibodies form the output of the third selection, wherein the one ormore antibodies comprise one or more light chains that each havespecificity for the first antigen and the second antigen; therebyobtaining or identifying the one or more common light chains. In certainembodiments, the first library comprises a naïve library; the secondlibrary comprises a naïve library; or the first library comprises anaïve library and the second library comprises a naïve library.

As will be understood by the artisan and as disclosed throughout, anantigen may comprise one or more epitopes. An “epitope” refers to aregion, structure, or motif on an antigen which is recognized by anantibody and to which the antibody binds. Thus, antibodies may beobtained or identified which have specificity for more than one epitopeon the same antigen. “Epitopic coverage”, “epitope coverage”, and liketerms refer to the extent to which such antibodies are collected,isolated, and/or identified when performing a selection or libraryinterrogation process that collectively have specificities for number ofepitopes that approximate the full repertoire, or diversity, ofavailable epitopes of the antigen of interest.

As will be understood by the artisan and as disclosed throughout,“specificity” refers to the property of an antibody which enables toreact with one or more antigenic determinants, such as one or moreepitopes, of an antigen of interest, and not with other epitopes of theantigen of interest or with other antigens of interest. As understood inthe art, antibody specificity is dependent on chemical composition,physical forces, energetic favorability, steric hindrance, and molecularstructure or topology of the binding site of the epitope and/or theantibody.

As will be understood by the artisan and as disclosed throughout,“affinity” refers to the strength, or stability of an antibody-epitopeinteraction. Antibodies with better affinity for an epitope bindrelatively tightly and/or stably with the epitope, whereas antibodieswith poorer affinity for an epitope bind relatively weakly and or lessstably. Although not always the case, it is often found that antibodieshaving greater specificity for an antigen of interest of an epitope ofinterest relative to another antibody has better affinity for thatantigen or epitope relative to the other antibody.

As will be understood by the artisan and as disclosed throughout,“obtaining” or “identifying” antibodies or components of antibodies(such as common light chains, heavy chains, or antigen binding regions)having specificity for (an) epitope(s) of an antigen of interest refersto distinguishing (or distinguished) antibodies that have suchspecificity from those antibodies that do not have such specificity.Obtaining or identifying antibodies having specificity for an antigen orepitope of interest need not require physical separation of antibodiesfrom those antibodies that do not have such specificity in order forthem to be distinguished. However, in certain embodiments, obtaining oridentifying antibodies having specificity for an antigen or epitope ofinterest comprises physically separating such antibodies from thoseantibodies that do not have such specificity. Exemplary methods andmeans for obtaining or identifying antibodies are known in the art, andinclude, for example, flow cytometry, florescence activated cell sorting(FACS), magnetic activated cell sorting (MACS), enzyme-linkedimmunosorbent assay (ELISA), and the like, and combinations thereof.

Any means for determining such specificity in the art may be employedfor determining such specificity in accordance with the methodsdisclosed throughout, and include, for example, labelling suchantibodies with a detectable label; detecting a detectable label;detecting a functional consequence of antibody binding to antigen orepitope on an antigen, such as competition with another antibody knownto have specificity for such epitope(s); modulation of protein-proteinor protein-ligand interaction between the antigen of interest and aknown protein interaction partner or ligand.

As will be understood by the artisan and as disclosed throughout,“plurality” and “pluralities” refer to, in the broadest sense, two ormore members of a group of items. In certain embodiments of theinvention some or all of the members of such a plurality may beessentially identical. In certain other embodiments of the invention,many, most, or all of the members of a plurality of items, while eachpossessing similar characteristics that merit their inclusion in theplurality, are nonetheless different in some discernible way and possessdifferent properties.

As will be appreciated by the artisan, the terms “plurality” and“library” (and “pluralities” and “libraries”) may be readily usedinterchangeably. However, in the context of the inventions disclosedthroughout, whereas a “plurality” of items, such as antibodies, nucleicacid encoding antibodies, or host cells, may comprise many or mostmembers that are essentially identical, a “library” of items, such asantibodies, nucleic acid encoding antibodies, or host cells comprisemembers many or most members that are unique.

In the context of antibodies that are employed in practicing thedisclosed inventions, a library (or plurality) of such antibodies willcomprise many or most members that each possess a unique primary acidsequence; however, such libraries (or pluralities) may also includemembers that have identical amino acid sequences. In certainembodiments, the variable regions of such members will comprise many ofthe differences in amino acid sequence between such members.

In the context of host cells that are employed in practicing thedisclosed inventions, a plurality (or library) of such host cells willcomprise host cell members, many of which that each express a uniqueantibody; however, such host cell pluralities (or libraries) may alsoinclude members that express identical antibody sequences. In certainembodiments, such host cells will also harbor nucleic acid thatcollectively encodes the antibody libraries that are collectivelyexpressed by the host cells.

As will be understood by the artisan and as disclosed throughout,“diversity” refers to a variety or a noticeable heterogeneity. The term“sequence diversity” refers to a variety of sequences which arecollectively representative of several possibilities of sequences, forexample, those found in natural human antibodies. For example, heavychain CDR3 (CDRH3) sequence diversity may refer to a variety ofpossibilities of combining the known human DH and H3-JH segments,including the N1 and N2 regions, to form heavy chain CDR3 sequences. Thelight chain CDR3 (CDRL3) sequence diversity may refer to a variety ofpossibilities of combining the naturally occurring light chain variableregion contributing to CDRL3 (i.e., L3-VL) and joining (i.e., L3-JL)segments, to form light chain CDR3 sequences. As used herein, H3-JHrefers to the portion of the IGHJ gene contributing to CDRH3. As usedherein, L3-VL and L3-JL refer to the portions of the IGLV and IGLJ genes(kappa or lambda) contributing to CDRL3, respectively.

As will be understood by the artisan and as disclosed throughoutantibody libraries suitable for use in accordance with the disclosedmethods may be designed and prepared by any method available in the artas disclosed, for example, in WO2009036379; WO2012009568; WO2010105256;U.S. Pat. No. 8,258,082; U.S. Pat. No. 6,300,064; U.S. Pat. No.6,696,248; U.S. Pat. No. 6,165,718; U.S. Pat. No. 6,500,644; U.S. Pat.No. 6,291,158; U.S. Pat. No. 6,291,159; U.S. Pat. No. 6,096,551; U.S.Pat. No. 6,368,805; U.S. Pat. No. 6,500,644; and the like.

It is often desirable to include one more maturation library selectionsas part of an antibody discovery process. Such maturation libraryselections, such as affinity maturation library selections, may beadvantageously incorporated into the methods disclosed herein. In suchembodiments the methods disclosed herein further comprise: a) performinga subsequent selection against the first antigen from a maturationlibrary; b) performing a subsequent selection against the second antigenfrom a maturation library; or c) performing a subsequent selectionagainst the first antigen from a maturation library and performing asubsequent selection against the second antigen from a maturationlibrary; after performing: a) step i; b) step ii; c) step iii; and/or d)step iv.

A “naive library” refers to a library of polynucleotides (orpolypeptides encoded by such polynucleotides) that has not beeninterrogated for the presence of antibodies having specificity aparticular antigen. A “naïve library” also refers to a library that isnot restricted to, or otherwise biased or enriched for, antibodysequences having specificity for any group of antigens, or for aparticular antigen. A naïve library is thus distinct from a “restrictedlibrary” and “maturation library (such as, for example, an “affinitymaturation library”), both of which are described below.

A naïve library may also comprise a “preimmune” library, which refers toa library that has sequence diversity and length diversity similar tonaturally occurring antibody sequences, such as human antibodysequences, before such naturally occurring sequences have undergonenegative selection and/or somatic hypermutation. Such preimmunelibraries may be designed and prepared so as to reflect or mimic thepre-immune repertoire, and/or may be designed and prepared based onrational design informed by the collection of human V, D, and J genes,and other large databases of human heavy and light chain sequences(e.g., publicly known germline sequences; sequences from Jackson et al,J. Immunol Methods, 2007, 324: 26, incorporated by reference in itsentirety; sequences from Lee et al., Immunogenetics, 2006, 57: 917,incorporated by reference in its entirety; and sequences compiled forrearranged VK and Vλ). Additional information may be found, for example,in Scaviner et al., Exp. Clin. Immunogenet, 1999, 16: 234; Tomlinson etal, J. Mol. Biol, 1992, 227: 799; and Matsuda et al, J. Exp. Med., 1998,188: 2151, each incorporated by reference in its entirety. In certainembodiments of the invention, cassettes representing the possible V, D,and J diversity found in the human repertoire, as well as junctionaldiversity (i.e., N1 and N2), are synthesized de novo as single ordouble-stranded DNA oligonucleotides. In certain embodiments of theinvention, oligonucleotide cassettes encoding CDR sequences areintroduced into yeast along with one or more acceptor vectors containingheavy or light chain chassis sequences. No primer-based PCRamplification or template-directed cloning steps from mammalian cDNA ormRNA are employed. Through standard homologous recombination, therecipient yeast recombines the cassettes (e.g., CDR3s) with the acceptorvector(s) containing the chassis sequence(s) and constant regions, tocreate a properly ordered synthetic, full-length human heavy chainand/or light chain immunoglobulin library that can be geneticallypropagated, expressed, displayed, and screened. One of ordinary skill inthe art will readily recognize that the chassis contained in theacceptor vector can be designed so as to produce constructs other thanfull-length human heavy chains and/or light chains. For example, incertain embodiments of the invention, the chassis may be designed toencode portions of a polypeptide encoding an antibody fragment orsubunit of an antibody fragment, so that a sequence encoding an antibodyfragment, or subunit thereof, is produced when the oligonucleotidecassette containing the CDR is recombined with the acceptor vector. Incertain embodiments, the invention provides a synthetic, preimmune humanantibody repertoire comprising about 107 to about 1020 antibody members,wherein the repertoire comprises:

(a) selected human antibody heavy chain chassis (i.e., amino acids 1 to94 of the heavy chain variable region, using Kabat's definition); (b) aCDRH3 repertoire, designed based on the human IGHD and IGHJ germlinesequences, the CDRH3 repertoire comprising the following:

(i) optionally, one or more tail regions;

(ii) one or more N1 regions, comprising about 0 to about 10 amino acidsselected from the group consisting of fewer than 20 of the amino acidtypes preferentially encoded by the action of terminal deoxynucleotidyltransferase (TdT) and functionally expressed by human B cells;

(iii) one or DH segments, based on one or more selected IGHD segments,and one or more N- or C-terminal truncations thereof;

(iv) one or more N2 regions, comprising about 0 to about 10 amino acidsselected from the group consisting of fewer than 20 of the amino acidspreferentially encoded by the activity of TdT and functionally expressedby human B cells; and (v) one or more H3-JH segments, based on one ormore IGHJ segments, and one or more N-terminal truncations thereof(e.g., down to XXWG); (c) one or more selected human antibody kappaand/or lambda light chain chassis; and

(d) a CDRL3 repertoire designed based on the human IGLV and IGLJgermline sequences, wherein “L” may be a kappa or lambda light chain.

Exemplary such preimmune libraries, and the design and composition ofpolynucleotide sequences (and polypeptide sequences encoded by them)comprising them, are further described in, for example, Lee et al.(Immunogenetics, 2006, 57: 917); Martin et al., Proteins, 1996, 25:130;WO 2009/036379; and WO 2012/09568.

A “maturation library” refers to a library that is designed to enhanceor improve at least one characteristic of an antibody sequence that isidentified upon interrogation of a library, such as a naïve library or apreimmune library, for the presence of antibody sequences havingspecificity for the antigen. Such maturation libraries may be generatedby incorporating nucleic acid sequences corresponding to: one or moreCDRs; one or more antigen binding regions; one or more VH or VL regions;and/or one or more heavy chains or light chains; obtained from oridentified in an interrogation of a naïve library (herein referred to as“antibody leads”) into libraries designed to further mutagenize in vitroor in vivo to generate libraries with diversity introduced in thecontext of the initial antibody leads. Such maturation libraries andmethods of making them are provided in, for example, WO 2009/036379 (forexample, at pages 75 through 77); and WO 2012/09568 (for example pages69 to 72), and include: maturation libraries in which variegation isperformed in which a CDRH3 of interest remains unaltered, and heavychain framework regions, CHRH1, and/or CHDH2 regions are variegated;libraries in which a CDRL3 of interest remains unaltered, and lightchain framework regions, CHRL1, and/or CHDL2 regions are variegated;libraries in which premade, diverse, light chains are combined with oneor more heavy chains of interest.

In certain embodiments of the invention, antibody libraries, whethernaïve libraries, maturation libraries, or restricted libraries, aredesigned to be small enough to chemically synthesize and physicallyrealize, but large enough to encode antibodies with the potential torecognize any antigen. In certain embodiments, an antibody librarycomprises about 10⁷ to about 10²⁰ different antibodies and/orpolynucleotide sequences encoding the antibodies of the library. In someembodiments, the libraries are designed to include 10³, 10⁴, 10⁵, 10⁶,10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸,10¹⁹, or 10²⁰ different antibodies and/or polynucleotide sequencesencoding the antibodies. In certain embodiments, the libraries maycomprise or encode about 10³ to about 10⁵, about 10⁵ to about 10⁷, about10⁷ to about 10⁹, about 10⁹ to about 10¹¹, about 10¹¹ to about 10¹³,about 10¹³ to about 10¹⁵, about 10¹⁵ to about 10¹⁷, or about 10¹⁷ toabout 10²⁰ different antibodies. In certain embodiments, the diversityof the libraries may be characterized as being greater than or less thanone or more of the diversities enumerated above, for example greaterthan about 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³,10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸, 10¹⁹, or 10²⁰ or less than about 10³, 10⁴,10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷,10¹⁸, 10¹⁹, or 10²⁰. In certain other embodiments of the invention, theprobability of an antibody of interest being present in a physicalrealization of a library with a size as enumerated above is at leastabout 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 5%, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 85%, 90%, 95%, 99%, 99.5%, or 99.9%

A “common light chain” refers to a an antibody light chain that: cancombine with a first heavy chain having specificity for a first antigen;and can combine with a second heavy chain having specificity for asecond antigen; such that an antigen binding region is formed which hasspecificity for the first antigen and an antigen binding region isformed which has specificity for the second antigen. Accordingly, amultispecific antibody analog may be generated comprising: the firstheavy chain; the second heavy chain; and the common light chain.

A “restricted library” refers to a library that comprises: one or moreunique heavy chains, one or more unique light chains, or one or moreunique heavy chains and one or more unique light chains; that have beenobtained or identified by performing a selection from, for example, anaive library for antigen binding regions having specificity for oneantigen of interest; and is used to obtain or identify antigen bindingregions having specificity for another antigen of interest. Suchrestricted libraries typically comprise a number of either heavy chainsor light chains that is in far excess of the number of light chains orheavy chains, respectively. In certain embodiments, the number of uniqueheavy chains is at least 10⁵, at least 10⁶, at least 10⁷, 10⁸, or atleast 10⁹ or greater and the number of unique light chains is one, two,three, four, five, ten, 15, 20, 50, 100, 200, 500, or 1000. In certainembodiments, the number of unique heavy chains is between 10⁷ and 10⁸,and the number of unique light chains is less than 10, preferablyapproximately 5.

Multispecific antibody analogs may also comprise antibodies comprise atleast two copies of a first polypeptide and at least two copies of asecond polypeptide, wherein each copy of the first polypeptide comprisesa variable heavy domain (VH) with specificity for one antigen ofinterest as well as a VH with specificity for a second antigen ofinterest; and wherein each copy of the second polypeptide comprises alight chain variable domain that is compatible with both VHs and canform a first antigen (also referred interchangeably throughout as an“antigen 1”) binding region and a second antigen (also referredinterchangeably throughout as an “antigen 2”) binding region whenassociated with the first VH and the second VH, respectively In certainof such embodiments, each copy of the second polypeptide comprises acopy of a common light chain. Accordingly, because such inventiveanalogs comprise two copies of one heavy chain-containing polypeptidespecies and two copies of one light chain-containing polypeptidespecies, such antibody analogs afford greatly streamlined productioncharacterized by greatly diminished, if not essentially eliminated,generation of undesired oligomeric species (i.e., homo dimeric heavychain specific, mis-pairing of light chains, etc.) that ischaracteristic of prior methods and analogs which require at least twodifferent VH polypeptide-containing specifies and/or at least two lightchain-containing polypeptide species as disclosed in, for example (see,e.g., U.S. Pat. No. 5,731,168; U.S. Pat. No. 5,807,706; U.S. Pat. No.5,821,333; U.S. Pat. No. 7,183,076; U.S. Pat. No. 7,642,228; U.S. Pat.No. 7,695,936; U.S. Ser. No. 11/536,951, U.S. Pat. No. 8,216,805, andU.S. Pat. No. 7,951,917). As a result, the herein disclosed and claimedanalogs and methods of their preparation afford correspondinglyaugmented yields of relatively pure amounts of the desired multispecificantibody analog.

The inventive methods by which the disclosed multispecific antibodyanalogs are generate comprise, for example: obtaining or identifying oneor more light chains from one or more first antigen binding regionshaving specificity for a first antigen of interest; performing a firstselection from, for example, a naïve library for antigen binding regionshaving specificity for a second antigen of interest; obtaining heavychains from the output of the selection against the second antigen andmixing them with one or more light chains thereby generating arestricted library; performing second selection against the secondantigen of interest with the restricted library; obtaining oridentifying from the output of restricted library selection one or moreantigen binding regions having specificity for the second antigen ofinterest, wherein the one or more antigen binding regions comprises theone or more light chains; and incorporating the antigen binding regionhaving specificity for the first antigen of interest and the one or moreantigen binding regions having specificity for the second antigen ofinterest into a multispecific antibody analog comprising: an IgG moietycomprising either:

a) the first antigen binding region; or

b) the second antigen binding region; and

two Fab moieties, wherein each Fab moiety comprises either:

a) the second antigen binding region; or

b) the first antigen binding region;

wherein the N-terminus of the heavy chain of one Fab moiety is linked tothe C-terminus of the Fc region of one heavy chain of the IgG moiety viaa linker moiety, and the N-terminus of the heavy chain of the other Fabmoiety is linked to the C-terminus of the Fc region of the other heavychain of the IgG moiety via a linker moiety; thereby generating themultispecific antibody analog.

In certain embodiments, the one or more light chains from the firstantigen binding regions is obtained or identified from the output of aselection performed with for example, a naïve library for antigenbinding regions against the first antigen of interest.

By “Fab” or “Fab region” as used herein is meant the polypeptides thatcomprise the VH, CH1, VL (also known as VK, used interchangeablythroughout), and CL (also known as CK, used interchangeably throughout)immunoglobulin domains. Typically, the VH and CH1 domains comprise onepolypeptide and the VL and CL domains comprise another polypeptide,wherein the two polypeptides are linked to one another via at least oneinter-polypeptide disulfide bond. Fab may refer to this region inisolation, or this region in the context of a full length antibody orantibody fragment.

By “protein” or “polypeptide” as used herein is meant at least twocovalently attached amino acids, which includes proteins, polypeptides,oligopeptides and peptides. The protein may be made up of naturallyoccurring amino acids and peptide bonds, or synthetic peptidomimeticstructures, i.e. “analogs”, such as peptides.

By “scFv” as used herein is meant a polypeptide consisting of twovariable regions connected by a linker sequence; e.g., VH-linker-VL,VH-linker-VL, Vκ-linker-VL, or VL-linker-VH. “Linkers” (also referred toa “linker moieties”, used interchangeably throughout), are described inmore detail below.

By “position” as used herein is meant a location in the sequence of aprotein or nucleic acid. Protein positions may be numbered sequentially,or according to an established format, for example the Kabat index forantibody variable regions or the EU index for antibody constant regions.For example, position 297 is a position in the human antibody IgG1.Corresponding positions are determined as outlined above, generallythrough alignment with other parent sequences.

By “residue” as used herein is meant a position in a protein and itsassociated amino acid identity. For example, Asparagine 297 (alsoreferred to as Asn297, also referred to as N297) is a residue in thehuman antibody IgG1. In some embodiments it can also refer to nucleicacid bases.

Antibodies (used interchangeably with “immunoglobulins”, or“immunoglobulin molecules”) can be monomeric, dimeric, trimeric,tetrameric, pentameric, etc., and comprise a class of structurallyrelated proteins consisting of two pairs of polypeptide chains: one pairof light chains (LC) and one pair of heavy chains (HC), all of which areinter-connected by disulfide bonds. The structure of immunoglobulins hasbeen well characterized. See for instance Fundamental Immunology Ch. 7(Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)).

Traditional natural antibody structural units typically comprise atetramer. Each tetramer is typically composed of two identical pairs ofpolypeptide chains, each pair having one “light” (typically having amolecular weight of about 25 kDa) and one “heavy” chain (typicallyhaving a molecular weight of about 50-70 kDa), which are referred toherein as a “light chain” and “heavy chain”, respectively. Human lightchains are classified as kappa and lambda light chains. Heavy chains areclassified as mu, delta, gamma, alpha, or epsilon, and define theantibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG hasseveral subclasses, including, but not limited to IgG1, IgG2, IgG3, andIgG4. IgM has subclasses, including, but not limited to, IgM1 and IgM2.IgA has several subclasses, including but not limited to IgA1 and IgA2.Thus, “isotype” as used herein is meant any of the classes andsubclasses of immunoglobulins defined by the chemical and antigeniccharacteristics of their constant regions. The known humanimmunoglobulin isotypes are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1,IgM2, IgD, and IgE. The distinguishing features between these antibodyclasses are their constant regions, although subtler differences mayexist in the variable region.

Each of the light and heavy chains is made up of two distinct regions,referred to as the variable and constant regions. The IgG heavy chain iscomposed of four immunoglobulin domains linked from N- to C-terminus inthe order VH-CH1-CH2-CH3, referring to the “variable heavy domain” (alsoreferred to as a “heavy chain variable domain”, used interchangeablythroughout), heavy chain constant domain 1, heavy chain constant domain2, and heavy chain constant domain 3 respectively (also referred to asVH-Cγ1-Cγ2-Cγ3, referring to the variable heavy domain, constant gamma 1domain, constant gamma 2 domain, and constant gamma 3 domainrespectively). The IgG light chain is composed of two immunoglobulindomains linked from N- to C-terminus in the order VL-CL, referring tothe “variable light domain” (also referred to as a “light chain variabledomain”, used interchangeably throughout) and the light chain constantdomain respectively. The constant regions show less sequence diversity,and are responsible for binding a number of natural proteins to elicitimportant biochemical events. The structure that constitutes the naturalbiological form of an antibody, including the variable and constantregions, is referred to herein as a “full length antibody”. In mostmammals, including humans and mice, the full length antibody of the IgGisotype is a tetramer and consists of two identical pairs of twoimmunoglobulin chains, each pair having one light chain and one heavychain, each light chain comprising a VL and a CL, and each heavy chaincomprising a VH, CH1, a CH2, and a CH3. In some mammals, for example incamels and llamas, IgG antibodies may consist of only two heavy chains,each heavy chain comprising a variable domain attached to the Fc region.

The heavy chain constant region typically is comprised of three domains,CH1, CH2, and CH3, and the CH1 and CH2 domains are connected by a hingeregion. Each light chain typically is comprised of a light chainvariable domain (abbreviated herein as “VL” or “VL”) and a light chainconstant domain. The VH and VL domains may be further subdivided intoregions of hypervariability (or hypervariable regions which may behypervariable in sequence and/or form of structurally defined loops),also termed complementarity determining regions (CDRs), interspersedwith regions that are more conserved, termed framework regions (FRs).Each VH and VL is typically composed of three CDRs and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Typically, the numbering of aminoacid residues in this region is performed by the method described inKabat (see, e.g., Kabat et al, in “Sequences of Proteins ofImmunological Interest,” 5th Edition, U.S. Department of Health andHuman Services, 1992). Using this numbering system, the actual linearamino acid sequence of a peptide may contain fewer or additional aminoacids corresponding to a shortening of, or insertion into, a FR or CDRof the variable domain. For example, a heavy chain variable domain mayinclude a single amino acid insert (residue 52a according to Kabat)after residue 52 of VH CDR2 and inserted residues (for instance residues82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue82. The Kabat numbering of residues may be determined for a givenantibody by alignment at regions of homology of the sequence of theantibody with a “standard” Kabat numbered sequence.

The term “variable”, “variable domain”, or “variable region” eachinterchangeably refers to the portions of the immunoglobulin domainsthat exhibit variability in their sequence and that are involved indetermining the specificity and binding affinity of a particularantibody (i.e., the “variable domain(s)”). Variability is not evenlydistributed throughout the variable domains of antibodies; it isconcentrated in sub-domains of each of the heavy and light chainvariable regions. These sub-domains are called “hypervariable” regionsor “complementarity determining regions” (CDRs). The more conserved(i.e., non-hypervariable) portions of the variable domains are calledthe “framework” regions (FRM). The variable domains of naturallyoccurring heavy and light chains each comprise four FRM regions, largelyadopting a β-sheet configuration, connected by three hypervariableregions, which form loops connecting, and in some cases forming part of,the β-sheet structure. The hypervariable regions in each chain are heldtogether in close proximity by the FRM and, with the hypervariableregions from the other chain, contribute to the formation of theantigen-binding site (see Kabat et al. Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md., 1991, incorporated by reference inits entirety). The constant domains are not directly involved in antigenbinding, but exhibit various effector functions, such as, for example,antibody-dependent, cell-mediated cytotoxicity and complementactivation.

The term “framework region” refers to the art-recognized portions of anantibody variable region that exist between the more divergent (i.e.,hypervariable) CDRs. Such framework regions are typically referred to asframeworks 1 through 4 (FRM1, FRM2, FRM3, and FRM4) and provide ascaffold for the presentation of the six CDRs (three from the heavychain and three from the light chain) in three dimensional space, toform an antigen-binding surface. The term “canonical structure” refersto the main chain conformation that is adopted by the antigen binding(CDR) loops. From comparative structural studies, it has been found thatfive of the six antigen binding loops have only a limited repertoire ofavailable conformations. Each canonical structure can be characterizedby the torsion angles of the polypeptide backbone. Correspondent loopsbetween antibodies may, therefore, have very similar three dimensionalstructures, despite high amino acid sequence variability in most partsof the loops (Chothia and Lesk, J. Mol. Biol., 1987, 196: 901; Chothiaet al, Nature, 1989, 342: 877; Martin and Thornton, J. Mol. Biol, 1996,263: 800. Furthermore, there is a relationship between the adopted loopstructure and the amino acid sequences surrounding it. The conformationof a particular canonical class is determined by the length of the loopand the amino acid residues residing at key positions within the loop,as well as within the conserved framework (i.e., outside of the loop).Assignment to a particular canonical class can therefore be made basedon the presence of these key amino acid residues.

The term “canonical structure” may also include considerations as to thelinear sequence of the antibody, for example, as catalogued by Kabat(Kabat et al, in “Sequences of Proteins of Immunological Interest,” 5thEdition, U.S. Department of Health and Human Services, 1992). The Kabatnumbering scheme is a widely adopted standard for numbering the aminoacid residues of an antibody variable domain in a consistent manner.Additional structural considerations can also be used to determine thecanonical structure of an antibody. For example, those differences notfully reflected by Kabat numbering can be described by the numberingsystem of Chothia et al and/or revealed by other techniques, forexample, crystallography and two or three-dimensional computationalmodeling. Accordingly, a given antibody sequence may be placed into acanonical class which allows for, among other things, identifyingappropriate chassis sequences (e.g., based on a desire to include avariety of canonical structures in a library). Kabat numbering ofantibody amino acid sequences and structural considerations as describedby Chothia et al., and their implications for construing canonicalaspects of antibody structure, are described in the literature.

By “Fc” or “Fc region”, as used herein is meant the polypeptidecomprising the constant region of an antibody excluding the firstconstant region immunoglobulin domain. Thus “Fc region” refers to thelast two constant region immunoglobulin domains of IgA, IgD, and IgG,and the last three constant region immunoglobulin domains of IgE andIgM, and the flexible hinge N-terminal to these domains. For IgA andIgM, Fc may include the J chain. For IgG, Fc comprises immunoglobulindomains Cgamma2 and Cgamma3 (Cγ2 and Cγ3) and the hinge between Cgamma1(Cγ1) and Cgamma2 (Cγ2). Accordingly, and without departing from theabove, “Fc region” may also be defined as comprising a “CH2 domain or avariant thereof” and a “CH3 domain or a variant thereof”. Although theboundaries of the Fc region may vary, the human IgG heavy chain Fcregion is usually defined to comprise residues C226 or P230 to itscarboxyl-terminus, wherein the numbering is according to the EU index asin Kabat. Fc may refer to this region in isolation, or this region inthe context of an Fc polypeptide, for example an antibody. By “Fcpolypeptide” as used herein is meant a polypeptide that comprises all orpart of an Fc region. Fc polypeptides include antibodies, Fc fusions,isolated Fcs, and Fc fragments.

A variable light chain (VL) and corresponding variable heavy domain (VH)of the inventive multispecific antibody analogs comprise a bindingdomain, also referred to interchangeably throughout as an “antigenbinding site” that interacts with an antigen. Such antigen binding sites(or binding regions, used interchangeably throughout), are optionallyprovided and employed in the context of either an IgG or a Fab of thepresent invention. Thus, a “first variable light domain” and a “firstvariable heavy domain” of the inventive multispecific antibody analogstogether form a “first antigen binding site”. Similarly, a “secondvariable light domain” and a “second variable heavy domain” of theinventive multispecific antibody analogs together form a “second antigenbinding site”. A “third variable light domain” and a “third variableheavy domain” of the inventive multispecific antibody analogs togetherform a “third antigen binding site”, and so on. Accordingly, theinventive multispecific antibody analogs may comprise an IgG comprisingtwo antigen binding regions, to which has been attached two Fabs,wherein such Fabs are each attached to the C-terminus of each CH3 regionof the IgG via a linker moiety.

The antigen binding sites for use in accordance with the invention,including the VHs, VLs, and/or CDRs that comprise such, may be obtainedor derived from any source of such, as will be understood by theartisan. Accordingly, such antigen binding sites, VHs, VLs, and/or CDRsmay be obtained or derived from hybridoma cells that express antibodiesagainst a target recognized by such; from B cells from immunized donors,which express antibodies against a target recognized by such; fromB-cells that have been stimulated to express antibodies against a targetrecognized by such; and or from identification of antibodies or antibodyfragments that have been identified by screening a library comprising aplurality of polynucleotides or polypeptides for antigen bindingantibodies (or antigen binding fragments thereof). With regard to thedesign, preparation, display, and implementation of such libraries foruse in identifying and obtaining antigen binding sites for use inaccordance with the invention, see, e.g., WO 2009/036379; WO2012009568;WO2010105256; U.S. Pat. No. 8,258,082; U.S. Pat. No. 6,300,064; U.S.Pat. No. 6,696,248; U.S. Pat. No. 6,165,718; U.S. Pat. No. 6,500,644;U.S. Pat. No. 6,291,158; U.S. Pat. No. 6,291,159; U.S. Pat. No.6,096,551; U.S. Pat. No. 6,368,805; U.S. Pat. No. 6,500,644; and thelike.

Any one or more of the antigen binding sites, VHs, VLs, or CDRs, andcombinations thereof, of the inventive multispecific antibody analogs,may comprise sequences from a variety of species. In some embodiments,such antigen binding sites, VHs, VLs, or CDRs, and combinations thereofmay be obtained from a nonhuman source, including but not limited tomice, rats, rabbits, camels, llamas, and monkeys. In some embodiments,the scaffold and/or framework regions can be a mixture from differentspecies. As such, a multispecific antibody analog in accordance with theinvention may comprise a chimeric antibody and/or a humanized antibody.In general, both “chimeric antibodies” and “humanized antibodies” referto antibodies in which regions from more than one species have beencombined. For example, “chimeric antibodies” traditionally comprisevariable region(s) from a mouse or other nonhuman species and theconstant region(s) from a human.

“Humanized antibodies” generally refer to non-human antibodies that havehad the variable-domain framework regions swapped for sequences found inhuman antibodies. Generally in a humanized antibody the entire antibody,except the CDRs, is encoded by a polynucleotide of human origin or isidentical to such an antibody except within its CDRs. The CDRs, one,some, or all of which are encoded by nucleic acids originating in anon-human organism, are grafted into the framework of a human antibodyvariable region to create an antibody, the specificity of which isdetermined by the engrafted CDRs. The creation of such antibodies isdescribed in, e.g., WO 92/11018, Jones, 1986, Nature 321:522-525,Verhoeyen et al., 1988, Science 239:1534-1536. “Backmutation” ofselected acceptor framework residues to the corresponding donor residuesis often required to regain affinity that is lost in the initial graftedconstruct (see, e.g., U.S. Pat. No. 5,693,762). The humanized antibodyoptimally also will comprise at least a portion of an immunoglobulinconstant region, typically that of a human immunoglobulin, and thus willtypically comprise a human Fc region. A variety of techniques andmethods for humanizing, reshaping, and resurfacing non-human antibodiesare well known in the art (See Tsurushita & Vasquez, 2004, Humanizationof Monoclonal Antibodies, Molecular Biology of B Cells, 533-545,Elsevier Science (USA), and references cited therein). In certainvariations, the immunogenicity of the antibody is reduced using a methoddescribed in Lazar et al., 2007, Mol Immunol 44:1986-1998 and U.S. Ser.No. 11/004,590, entitled “Methods of Generating Variant Proteins withIncreased Host String Content and Compositions Thereof”, filed on Dec.3, 2004.

Accordingly, any one or more of the antigen binding sites, or one ormore VHs, VLs, CDRs, or combinations thereof, which comprise theinventive multispecific antibody analogs disclosed herein may be derivedfrom a non-human species and/or result from humanization of a non-humanantibody or antibody fragment. Such VHs, VLs, and/or CDRs obtained orderived from non-human species, when included in the inventivemultispecific antibody analogs disclosed herein, are referred to as“humanized” such regions and/or domains.

The inventive antibody analogs disclosed herein preferably comprisefirst and second polypeptides that each comprise a hinge region, whereineach hinge region comprises at least one thiol group that is capable ofparticipating in an intermolecular disulfide bond such that the firstand the second polypeptide are covalently linked as a result offormation of the disulfide bond. As is understood in the art, chemicalmodification may be introduced into (or onto) certain residues withinsuch hinge regions which effect the introduction of such thiol groupsfor disulfide bond formation. Alternatively, the thiol groups may beprovided by a cysteine residue that is present within the hinge region.Such cysteines may be provided by native hinge polypeptide sequence, ormay be introduced by mutagenesis into nucleic acid encoding the hingeregion. As used herein, whereas a “hinge” or a “hinge region” of theinventive antibody analogs may comprise or constitute a natural ornative hinge region as found in, for example, immunoglobulins such asIgGs, IgMs, IgAs, IgEs, and the like, such a hinge or hinge region mayalso comprise or constitute a substitutes form thereof. Further, such ahinge or hinge region may, in certain embodiments comprise or constitutea “linker moiety” as disclosed throughout. In other embodiments, a hingeor hinge region may comprise both a natural or native hinge region asdisclosed above and a linker moiety as disclosed throughout.

In certain embodiments, the inventive antibody analogs disclosed hereincomprise one or more linkers or linker moieties. Such linkers or linkermoieties may comprise a peptidic linker moiety or a non-peptidic linkermoiety. The terms “linker” and “linker moiety” and the like, means adivalent species (-L-) covalently bonded in turn to a polypeptide havinga valency available for bonding and to an amino acid that comprises theinventive multispecific antibody analogs, which amino acid has a valencyavailable for bonding. The available bonding site may convenientlycomprise a side chain of an amino acid (e.g., a lysine, cysteine, oraspartic acid side chain, and homologs thereof). In some embodiments,the available bonding site in the analog is the side chain of a lysineor a cysteine residue. In some embodiments, the available bonding sitein the analog is the N-terminal amine of a polypeptide comprising theanalog. In some embodiments, the available bonding site in the analog isthe C-terminal carboxyl of a polypeptide comprising the analog. In someembodiments, the available bonding site in the analog is a backbone atom(e.g., a c-alpha carbon atom) of a polypeptide comprising the analog.

Preferably, a linker moiety is employed to covalently attach a VH or aVL to the C-terminus of a CH3 domain of an antibody analog. A linkermoiety may also be employed to covalently attach a first VH or a firstVL to a second VH or a second VL, respectively. A linker moiety may alsobe employed to covalently attach a first VH or a first VL to a second VLor a second VH, respectively. A linker moiety may also be employed tocovalently attach a VH of a single chain antigen binding site, such asan scFv, to the VL of such a single chain antigen binding site, and viceversa. A linker moiety may also be employed to attach the VH or the VLof such a single chain antigen binding site, such as an scFv, to aC-terminus of a CH3 domain or variant thereof. A linker moiety may alsobe employed to attach a VH to the N-terminus of a CL domain or to theN-terminus of a CH2. A linker moiety may also be employed to attach a VLto the N-terminus of a CL domain or to the N-terminus of a CH2 domain.As will be appreciated, combinations and/or multiples of the foregoingmay be employed in order to prepare any of the multispecific antibodyanalogs disclosed herein, such that a plurality of antigen binding sitesmay be included in such analogs, optionally with a multiple ofspecificities. Accordingly, a multispecific antibody analog may begenerated by employing one or more linkers to covalently attach one,two, three, four, five, six, seven, or more VLs, VHs, and/or singlechain antigen binding sites, such as scFvs to the first polypeptide, thesecond polypeptide, a VH, or a VL attached to the first polypeptide orthe second polypeptide, and the like, so as to generate an antibodyanalog having bi-, tri-, tetra-, pent-, hexa-, hepta-, or octa-valency,and so on, and/or bi-, tri-, tetra-, pent-, hexa-, hepta-, orocta-specificity, and so on.

In certain embodiments, the VH region of a Fab is attached to the CH3region of each heavy chain of an IgG in order to generate the inventivemultispecific antibody analogs.

In certain embodiments the linker moieties comprise amino acids that areselected from glycine, alanine, proline, asparagine, glutamine, lysine,aspartate, and glutamate. In a further embodiment the linker moiety ismade up of a majority of amino acids that are sterically unhindered,such as glycine, alanine and/or serine. In certain embodiments thelinker moiety is comprises a sequence selected from the group Gly-Ser]n(SEQ ID NO: 8); [Gly-Gly-Ser]n (SEQ ID NO: 9); [Gly-Gly-Gly-Ser]n (SEQID NO: 10); [Gly-Gly-Gly-Gly-Ser]n (SEQ ID NO: 11);[Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly]n (SEQ ID NO: 12);[Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly]n (SEQ ID NO:13); [Gly-Gly-Gly-Gly-SerGly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly]n (SEQ ID NO:14);[Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly]n(SEQ ID NO: 15);[Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly]n(SEQ ID NO: 16);[Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly]n(SEQ ID NO: 17); and combinations thereof; where n is an integerselected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, and 75.

Such linkers may comprise: an acidic linker, a basic linker, and astructural motif, or combinations thereof; a polyglycine, a polyalanine,poly(Gly-Ala), or poly(Gly-Ser); (Gly)3 (SEQ ID NO: 1), (Gly)4 (SEQ IDNO: 2), or (Gly)5 (SEQ ID NO: 3); (Gly)3Lys(Gly)4 (SEQ ID NO: 4),(Gly)3AsnGlySer(Gly)2 (SEQ ID NO: 5), (Gly)3Cys(Gly)4 (SEQ ID NO: 6), orGlyProAsnGlyGly (SEQ ID NO: 7), [Gly-Ser]n (SEQ ID NO: 8),[Gly-Gly-Ser]n (SEQ ID NO: 9), [Gly-Gly-Gly-Ser]n (SEQ ID NO: 10),[Gly-Gly-Gly-Gly-Ser]n (SEQ ID NO: 11),[Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly]n (SEQ ID NO: 12),[Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly]n (SEQ ID NO:13), [Gly-Gly-Gly-Gly-SerGly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly]n (SEQ ID NO:14),[Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly]n(SEQ ID NO: 15),[Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly]n(SEQ ID NO: 16), or[Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly]n(SEQ ID NO: 17); [Gly-Glu]n (SEQ ID NO: 18), [Gly-Gly-Glu]n (SEQ ID NO:19), [Gly-Gly-Gly-Glu]n (SEQ ID NO: 20), [Gly-Gly-Gly-Gly-Glu]n (SEQ IDNO: 21), [Gly-Asp]n (SEQ ID NO: 22); [Gly-Gly-Asp]n (SEQ ID NO: 23),[Gly-Gly-Gly-Asp]n (SEQ ID NO: 24), [Gly-Gly-Gly-Gly-Asp]n (SEQ ID NO:25); where n is an integer selected from the group consisting of 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, and 75.

In certain embodiments, charged linker moieties are employed. Suchcharges linker moieties may contain a significant number of acidicresidues (e.g., Asp, Glu, and the like), or may contain a significantnumber of basis residues (e.g., Lys, Arg, and the like), such that thelinker moiety has a pi lower than 7 or greater than 7, respectively. Asunderstood by the artisan, and all other things being equal, the greaterthe relative amount of acidic or basic residues in a given linkermoiety, the lower or higher, respectively, the pI of the linker moietywill be. Such linker moieties may impart advantages to the multispecificantibody analogs disclosed herein, such as improving solubility and/orstability characteristics of such polypeptides at a particular pH, suchas a physiological pH (e.g., between H 7.2 and pH 7.6, inclusive), or apH of a pharmaceutical composition comprising such analogs, as well asallowing for optimization of characteristics such as rotational andtranslational flexibility of the domains and/or regions of the analogthat are attached via the linker moiety. Such characteristics mayadvantageously be optimized and tailored for any given multispecificantibody analog by the artisan.

For example, an “acidic linker” is a linker moiety that has a pI of lessthan 7; between 6 and 7, inclusive; between 5 and 6, inclusive; between4 and 5, inclusive; between 3 and 4, inclusive; between 2 and 3,inclusive; or between 1 and 2, inclusive. Similarly, a “basic linker” isa linker moiety that has a pI of greater than 7; between 7 and 8,inclusive; between 8 and 9, inclusive; between 9 and 10, inclusive;between 10 and 11, inclusive; between 11 and 12 inclusive, or between 12and 13, inclusive. In certain embodiments, an acidic linker will containa sequence that is selected from the group consisting of [Gly-Glu]n (SEQID NO: 18); [Gly-Gly-Glu]n (SEQ ID NO: 19); [Gly-Gly-Gly-Glu]n (SEQ IDNO: 20); [Gly-Gly-Gly-Gly-Glu]n (SEQ ID NO: 21); [Gly-Asp]n (SEQ ID NO:22); [Gly-Gly-Asp]n (SEQ ID NO: 23); [Gly-Gly-Gly-Asp]n (SEQ ID NO: 24);[Gly-Gly-Gly-Gly-Asp]n (SEQ ID NO: 25); and combinations thereof; wheren is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, and 75. In certainembodiments, a basic linker will contain a sequence that is selectedfrom the group consisting of [Gly-Lys]; [Gly-Gly-Lys]n (SEQ ID NO: 83);[Gly-Gly-Gly-Lys]n (SEQ ID NO: 84); [Gly-Gly-Gly-Gly-Lys]n (SEQ ID NO:85); [Gly-Arg]n (SEQ ID NO: 86); [Gly-Gly-Arg]n (SEQ ID NO: 87);[Gly-Gly-Gly-Arg]n (SEQ ID NO: 88); [Gly-Gly-Gly-Gly-Arg]n (SEQ ID NO:89); and combinations thereof; where n is an integer selected from thegroup consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, and 75.

Additionally, linker moieties may be employed which possess certainstructural motifs or characteristics, such as an alpha helix. Forexample, such a linker moiety may contain a sequence that is selectedfrom the group consisting of [Glu-Ala-Ala-Ala-Lys]n (SEQ ID NO: 90),where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, and 75: for example, [Glu-Ala-Ala-Ala-Lys]3 (SEQ ID NO: 91),[Glu-Ala-Ala-Ala-Lys]4 (SEQ ID NO: 92), or [Glu-Ala-Ala-Ala-Lys]5 (SEQID NO: 93), and so on.

In still further embodiments the each linker moiety employed in thedisclosed multispecific antibody analogs independently comprises:polyglycine, polyalanine, poly(Gly-Ala), or poly(Gly-Ser), (Gly)3 (SEQID NO: 1), (Gly)4 (SEQ ID NO: 2), and (Gly)5 (SEQ ID NO: 3),(Gly)3Lys(Gly)4 (SEQ ID NO: 4), (Gly) 3AsnGlySer(Gly)2 (SEQ ID NO: 5),(Gly)3Cys(Gly)4 (SEQ ID NO: 6), and GlyProAsnGlyGly (SEQ ID NO: 7), acombination of Gly and Ala, a combination of Gly and Ser, a combinationof, Gly and Glu, a combination of Gly and Asp, a combination of Gly andLys, or combinations thereof.

It is understood in the art that, for many prior multispecific antibodyanalogs and methods of preparing them, there has been a requirement toengineer or design certain motifs into the Fc region, for example theCH3 domain of heavy chains in order to favor heterodimerization ofdifferent heavy chains—as opposed to homodimerization of identical heavychains—in order to generate meaningful quantities and purities of thedesired multispecific analog (see, e.g., U.S. Pat. No. 5,731,168; U.S.Pat. No. 5,807,706; U.S. Pat. No. 5,821,333; U.S. Pat. No. 7,183,076;U.S. Pat. No. 7,642,228; U.S. Pat. No. 7,695,936; U.S. Ser. No.11/536,951, U.S. Pat. No. 8,216,805, and U.S. Pat. No. 7,951,917). Suchengineering, while burdensome, may also introduce untoward antigenicityand/or immunogenicity characteristics to any potential antibody-basedtherapeutic, as well as other downstream formulation complications thatmay not be realized until relatively late in development.Advantageously, the herein disclosed and claims multispecific antibodyanalogs, and methods of their making, eliminate the requirement toengineer such heterodimerization motifs, and thus negate such concernsor complications. Indeed, because the VH domains for two or more targets(antigens) of interest are present on the same polypeptide in the hereindisclosed analogs, there is only one VH-containing polypeptide thatneeds to be expressed, along with a single light chain specific.Accordingly, as there are only two different species of polypeptidechains that are expressed and that are required to associate in order togenerate the inventive multispecific antibody analogs, the generation ofcontaminating, undesired oligomeric species is almost non-existent, orat least greatly diminished. Accordingly, there exists essentially onlyone four-chain specifies that is possible to be formed in accordancewith the herein disclosed methods, and the natural dimerization motifsfound in the Fc, CH1-CK, and VH-VL domains are sufficient to promoteefficient yield and recovery of properly associated and properly folded,four-chain (two heavy chain-containing, two light chain-containing)comprising multispecific antibody analogs as disclosed and claimedherein.

Thus, whereas it is understood that the inventive multispecific antibodyanalogs do not require the design or engineering of heterodimerizationmotifs in order to obtain meaningful quantities and purities of thedesired analog, the methods and analogs disclosed herein are nonethelessamenable to the inclusion of such motifs. In certain embodiments, theinventive multispecific antibody analogs comprise, for example, a CH2domain variant and/or a CH3 domain variant, wherein such variants eachindependently comprise at least one different amino acid substitutionsuch that a heterodimeric domain pair is generated such thatheterodimerization of the first and second polypeptides of the inventivemultispecific antibody analogs favored over homodimerization.

With regard to a “variant” of a domain or region of a multispecificantibody analog as used herein throughout, such a variant refers apolypeptide sequence that comprises such a domain or region, and thatdiffers from that of a parent polypeptide sequence by virtue of at leastone amino acid modification. The parent polypeptide sequence may be anaturally occurring or wild-type (WT) polypeptide sequence, or may be amodified version of a WT sequence. Preferably, the variant has at leastone amino acid modification compared to the parent polypeptide, region,or domain, e.g. from about one to about ten amino acid modifications,and preferably from about one to about five amino acid modificationscompared to the parent. The variant polypeptide sequence herein willpreferably possess at least about 80% homology with a parent sequence,and most preferably at least about 90% homology, more preferably atleast about 95% homology.

By “parent polypeptide”, “parent polypeptide sequence”, “parentprotein”, “precursor polypeptide”, or “precursor protein” as used hereinis meant an unmodified polypeptide or polypeptide sequence that issubsequently modified to generate a variant polypeptide or polypeptidesequence. Said parent polypeptide may be a naturally occurringpolypeptide, or a variant or engineered version of a naturally occurringpolypeptide. Parent polypeptide may refer to the polypeptide itself,compositions that comprise the parent polypeptide, or the amino acidsequence that encodes it.

By “Fc variant” or “variant Fc” as used herein is meant an Fc sequencethat differs from that of a parent Fc sequence by virtue of at least oneamino acid modification. An Fc variant may only encompass an Fc region,or may exist in the context of an antibody, Fc fusion, isolated Fc, Fcfragment, or other polypeptide that is substantially encoded by Fc. Fcvariant may refer to the Fc polypeptide itself, compositions comprisingthe Fc variant polypeptide, or the amino acid sequence that encodes it.

By “Fc polypeptide variant” or “variant Fc polypeptide” as used hereinis meant an Fc polypeptide that differs from a parent Fc polypeptide byvirtue of at least one amino acid modification. By “Fc variant antibody”or “antibody Fc variant” as used herein is meant an antibody thatdiffers from a parent antibody by virtue of at least one amino acidmodification in the Fc region.

By “protein variant” or “variant protein” as used herein is meant aprotein that differs from a parent protein by virtue of at least oneamino acid modification. By “antibody variant” or “variant antibody” asused herein is meant an antibody that differs from a parent antibody byvirtue of at least one amino acid modification. By “IgG variant” or“variant IgG” as used herein is meant an antibody that differs from aparent IgG by virtue of at least one amino acid modification. By“immunoglobulin variant” or “variant immunoglobulin” as used herein ismeant an immunoglobulin sequence that differs from that of a parentimmunoglobulin sequence by virtue of at least one amino acidmodification.

As mentioned above, although the inventive multispecific antibodyanalogs do not require the design or engineering of heterodimerizationmotifs in order to obtain meaningful quantities and purities of thedesired analog, the methods and analogs disclosed herein are nonethelessamenable to the inclusion of such motifs. Interaction betweenheterodimeric pairs or disclosed multispecific antibody analogscomprising such heterodimeric pairs may be promoted at the heterodimericpair interface by the formation of protuberance-into-cavitycomplementary regions at such interfaces; the formation of non-naturallyoccurring disulfide bonds at such interfaces; leucine zipper at suchinterfaces; hydrophobic regions at such interfaces; and/or hydrophilicregions at such interfaces. “Protuberances” are constructed by replacingsmall amino acid side chains from the interface of the first polypeptidewith larger side chains (e.g. tyrosine or tryptophan). Compensatory“cavities” of identical or similar size to the protuberances areoptionally created on the interface of the second polypeptide byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). Where a suitably positioned and dimensioned protuberanceor cavity exists at the interface of either the first or secondpolypeptide, it is only necessary to engineer a corresponding cavity orprotuberance, respectively, at the adjacent interface. Non-naturallyoccurring disulfide bonds are constructed by replacing on the firstpolypeptide a naturally occurring amino acid with a freethiol-containing residue, such as cysteine, such that the free thiolinteracts with another free thiol-containing residue on the secondpolypeptide such that a disulfide bond is formed between the first andsecond polypeptides. Exemplary heterodimerization pairs and methods formaking such in accordance with the present invention are available inthe art, and are disclosed, for example, in US 2011/0054151; US2007/0098712; and the like.

In certain embodiments, the heterodimeric pairs are contained within theFc region of the inventive multispecific antibody analogs. Fc regionsthat contain such heterodimeric pairs are referred to as “heterodimericFc regions”.

Accordingly, in certain embodiments, multispecific antibody analogscomprise a CH2 and/or a CH3 domain variant, wherein either: a) the CH2domain variant and the CH3 domain variant each independently comprises aat least one protuberance in either the CH2 domain or the CH3 domain ofthe first polypeptide and at least one corresponding cavity in the CH2domain or the CH3 domain of the second; or the CH2 domain variant andthe CH3 domain variant each independently comprises at least one cavityin either the CH2 domain or the CH3 domain of the first polypeptide andat least one corresponding protuberance in the CH2 domain or the CH3domain of the second polypeptide. In certain other embodiments, themultispecific antibody analogs comprise a CH2 and/or a CH3 domainvariant, wherein either: a) the CH2 domain variant and the CH3 domainvariant each independently comprises at least one substitutednegatively-charged amino acid in either the CH2 domain or the CH3 domainof the first polypeptide and at least one correspondingpositively-charged amino acid in either the CH2 domain or the CH3 domainof the second polypeptide; or b) the CH2 domain variant and the CH3domain variant each independently comprises at least one substitutedpositively-charged amino acid in either the CH2 domain or the CH3 domainof the first polypeptide and at least one corresponding substitutednegatively-charged substituted amino acid in either the CH2 domain orthe CH3 domain of the second polypeptide.

With regard to Fc function in “natural” antibodies (i.e., thoseantibodies generated in vivo via native biological antibody synthesis bynative B-cells), the Fc region of an antibody interacts with a number ofFc receptors and ligands, imparting an array of important functionalcapabilities referred to as effector functions. For IgG the Fc region,Fc comprises Ig domains Cγ2 and Cγ3 and the N-terminal hinge leadinginto Cγ2. An important family of Fc receptors for the IgG class is theFc gamma receptors (FcγRs). These receptors mediate communicationbetween antibodies and the cellular arm of the immune system (Raghavanet al., 1996, Annu Rev Cell Dev Biol 12:181-220; Ravetch et al., 2001,Annu Rev Immunol 19:275-290). In humans this protein family includesFcγRI (CD64), including isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII(CD32), including isoforms FcγRIIa (including allotypes H131 and R131),FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII(CD16), including isoforms FcγRIIIa (including allotypes V158 and F158)and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2)(Jefferis et al., 2002, Immunol Lett 82:57-65). These receptorstypically have an extracellular domain that mediates binding to Fc, amembrane spanning region, and an intracellular domain that may mediatesome signaling event within the cell. These receptors are expressed in avariety of immune cells including monocytes, macrophages, neutrophils,dendritic cells, eosinophils, mast cells, platelets, B cells, largegranular lymphocytes, Langerhans' cells, natural killer (NK) cells, andγδ T cells. Formation of the Fc/FcγR complex recruits these effectorcells to sites of bound antigen, typically resulting in signaling eventswithin the cells and important subsequent immune responses such asrelease of inflammation mediators, B cell activation, endocytosis,phagocytosis, and cytotoxic attack. The ability to mediate cytotoxic andphagocytic effector functions is a potential mechanism by whichantibodies destroy targeted cells. The cell-mediated reaction whereinnonspecific cytotoxic cells that express FcγRs recognize bound antibodyon a target cell and subsequently cause lysis of the target cell isreferred to as antibody dependent cell-mediated cytotoxicity (ADCC)(Raghavan et al., 1996, Annu Rev Cell Dev Biol 12:181-220; Ghetie etal., 2000, Annu Rev Immunol 18:739-766; Ravetch et al., 2001, Annu RevImmunol 19:275-290). The cell-mediated reaction wherein nonspecificcytotoxic cells that express FcγRs recognize bound antibody on a targetcell and subsequently cause phagocytosis of the target cell is referredto as antibody dependent cell-mediated phagocytosis (ADCP).

The different IgG subclasses have different affinities for the FcγRs,with IgG1 and IgG3 typically binding substantially better to thereceptors than IgG2 and IgG4 (Jefferis et al., 2002, Immunol Lett82:57-65). The FcγRs bind the IgG Fc region with different affinities.The extracellular domains of FcγRIIIa and FcγRIIIb are 96% identical;however FcγRIIIb does not have a intracellular signaling domain.Furthermore, whereas FcγRI, FcγRIIa/c, and FcγRIIIa are positiveregulators of immune complex-triggered activation, characterized byhaving an intracellular domain that has an immunoreceptor tyrosine-basedactivation motif (ITAM), FcγRIIb has an immunoreceptor tyrosine-basedinhibition motif (ITIM) and is therefore inhibitory. Thus the former arereferred to as activation receptors, and FcγRIIb is referred to as aninhibitory receptor. Despite these differences in affinities andactivities, all FcγRs bind the same region on Fc, at the N-terminal endof the Cγ2 domain and the preceding hinge.

An overlapping but separate site on Fc serves as the interface for thecomplement protein C1q. In the same way that Fc/FcγR binding mediatesADCC, Fc/C1q binding mediates complement dependent cytotoxicity (CDC). Asite on Fc between the Cγ2 and Cγ3 domains mediates interaction with theneonatal receptor FcRn, the binding of which recycles endocytosedantibody from the endosome back to the bloodstream (Raghavan et al.,1996, Annu Rev Cell Dev Biol 12:181-220; Ghetie et al., 2000, Annu RevImmunol 18:739-76). This process, coupled with preclusion of kidneyfiltration due to the large size of the full length molecule, results infavorable antibody serum half-lives ranging from one to three weeks.Binding of Fc to FcRn also plays a key role in antibody transport. Thebinding site for FcRn on Fc is also the site at which the bacterialproteins A and G bind. The tight binding by these proteins is typicallyexploited as a means to purify antibodies by employing protein A orprotein G affinity chromatography during protein purification. Thefidelity of these regions, the complement and FcRn/protein A bindingregions are important for both the clinical properties of antibodies andtheir development.

A particular feature of the Fc region of “natural” antibodies is theconserved N-linked glycosylation that occurs at N297. This carbohydrate,or oligosaccharide as it is sometimes referred, plays a criticalstructural and functional role for the antibody, and is one of theprinciple reasons that antibodies must be produced using mammalianexpression systems. Efficient Fc binding to FcγR and C1q requires thismodification, and alterations in the composition of the N297carbohydrate or its elimination affect binding to these proteins.

In some embodiments, the inventive multispecific antibody analogsdisclosed herein comprise an Fc variant. An Fc variant comprises one ormore amino acid modifications relative to a parent Fc polypeptide,wherein the amino acid modification(s) provide one or more optimizedproperties. Fc variants further comprise either a CH2 domain variant, aCH3 domain variant, or both a CH2 domain variant and a CH3 domainvariant. By “modification” herein is meant an alteration in thephysical, chemical, or sequence properties of a protein, polypeptide,antibody, inventive multispecific antibody analog, or immunoglobulin. Anamino acid modification can be an amino acid substitution, insertion,and/or deletion in a polypeptide sequence. By “amino acid substitution”or “substitution” herein is meant the replacement of an amino acid at aparticular position in a parent polypeptide sequence with another aminoacid. For example, the substitution Y349T refers to a variantpolypeptide, in this case a constant heavy chain variant, in which thetyrosine at position 349 is replaced with threonine. By “amino acidinsertion” or “insertion” as used herein is meant the addition of anamino acid at a particular position in a parent polypeptide sequence. By“amino acid deletion” or “deletion” as used herein is meant the removalof an amino acid at a particular position in a parent polypeptidesequence.

An Fc variant disclosed herein differs in amino acid sequence from itsparent by virtue of at least one amino acid modification. The inventivemultispecific antibody analogs disclosed herein may have more than oneamino acid modification as compared to the parent, for example fromabout one to fifty amino acid modifications, e.g., from about one to tenamino acid modifications, from about one to about five amino acidmodifications, etc. compared to the parent. Thus the sequences of the Fcvariants and those of the parent Fc polypeptide are substantiallyhomologous. For example, the variant Fc variant sequences herein willpossess about 80% homology with the parent Fc variant sequence, e.g., atleast about 90% homology, at least about 95% homology, at least about98% homology, at least about 99% homology, etc. Modifications disclosedherein also include glycoform modifications. Modifications may be madegenetically using molecular biology, or may be made enzymatically orchemically.

Fc variants disclosed herein are defined according to the amino acidmodifications that compose them. Thus, for example, the substitutionY349T refers to a variant polypeptide, in this case a constant heavychain variant, in which the tyrosine at position 349 is replaced withthreonine. Likewise, Y349T/T394F defines an Fc variant with thesubstitutions Y349T and T394F relative to the parent Fc polypeptide. Theidentity of the WT amino acid may be unspecified, in which case theaforementioned variant is referred to as 349T/394F. It is noted that theorder in which substitutions are provided is arbitrary, that is to saythat, for example, 349T/394F is the same Fc variant as 394F/349T. Unlessotherwise noted, constant region and Fc positions discussed herein arenumbered according to the EU index or EU numbering scheme (Kabat et al.,1991, Sequences of Proteins of Immunological Interest, 5th Ed., UnitedStates Public Health Service, National Institutes of Health, Bethesda).The EU index or EU index as in Kabat or EU numbering scheme refers tothe numbering of the EU antibody (Edelman et al., 1969, Proc Natl AcadSci USA 63:78-85).

In certain embodiments, the Fc variants disclosed herein are based onhuman IgG sequences, and thus human IgG sequences are used as the “base”sequences against which other sequences are compared, including but notlimited to sequences from other organisms, for example rodent andprimate sequences. Immunoglobulins may also comprise sequences fromother immunoglobulin classes such as IgA, IgE, IgD, IgM, and the like.It is contemplated that, although the Fc variants disclosed herein areengineered in the context of one parent IgG, the variants may beengineered in or “transferred” to the context of another, second parentIgG. This is done by determining the “equivalent” or “corresponding”residues and substitutions between the first and second IgG, typicallybased on sequence or structural homology between the sequences of thefirst and second IgGs. In order to establish homology, the amino acidsequence of a first IgG outlined herein is directly compared to thesequence of a second IgG. After aligning the sequences, using one ormore of the homology alignment programs known in the art (for exampleusing conserved residues as between species), allowing for necessaryinsertions and deletions in order to maintain alignment (i.e., avoidingthe elimination of conserved residues through arbitrary deletion andinsertion), the residues equivalent to particular amino acids in theprimary sequence of the first immunoglobulin are defined. Alignment ofconserved residues may conserve 100% of such residues. However,alignment of greater than 75% or as little as 50% of conserved residuesis also adequate to define equivalent residues. Equivalent residues mayalso be defined by determining structural homology between a first andsecond IgG that is at the level of tertiary structure for IgGs whosestructures have been determined. In this case, equivalent residues aredefined as those for which the atomic coordinates of two or more of themain chain atoms of a particular amino acid residue of the parent orprecursor (N on N, CA on CA, C on C and 0 on 0) are within about 0.13nm, after alignment. In another embodiment, equivalent residues arewithin about 0.1 nm after alignment. Alignment is achieved after thebest model has been oriented and positioned to give the maximum overlapof atomic coordinates of non-hydrogen protein atoms of the proteins.Regardless of how equivalent or corresponding residues are determined,and regardless of the identity of the parent IgG in which the IgGs aremade, what is meant to be conveyed is that the Fc variants discovered asdisclosed herein may be engineered into any second parent IgG that hassignificant sequence or structural homology with the Fc variant. Thusfor example, if a variant antibody is generated wherein the parentantibody is human IgG1, by using the methods described above or othermethods for determining equivalent residues, the variant antibody may beengineered in another IgG1 parent antibody that binds a differentantigen, a human IgG2 parent antibody, a human IgA parent antibody, amouse IgG2a or IgG2b parent antibody, and the like. Again, as describedabove, the context of the parent Fc variant does not affect the abilityto transfer the Fc variants disclosed herein to other parent IgGs.

Fc variants that comprise or are CH3 domain variants as described abovemay comprise at least one substitution at a position in a CH3 domainselected from the group consisting of 349, 351, 354, 356, 357, 364, 366,368, 370, 392, 394, 395, 396, 397, 399, 401, 405, 407, 409, 411, and439, wherein numbering is according to the EU index as in Kabat. In apreferred embodiment, CH3 domain variants comprise at least one CH3domain substitution per heavy chain selected from the group consistingof 349A, 349C, 349E, 349I, 349K, 349S, 349T, 349W, 351 E, 351K, 354C,356K, 357K, 364C, 364D, 364E, 364F, 364G, 364H, 364R, 364T, 364Y, 366D,366K, 366S, 366W, 366Y, 368A, 368E, 368K, 368S, 370C, 370D, 370E, 370G,370R, 370S, 370V, 392D, 392E, 394F, 394S, 394W, 394Y, 395T, 395V, 396T,397E, 397S, 397T, 399K, 401 K, 405A, 405S, 407T, 407V, 409D, 409E, 411D, 411 E, 411K, and 439D. Each of these variants can be usedindividually or in any combination for each heavy chain Fc region. Aswill be appreciated by those in the art, each heavy chain can comprisedifferent numbers of substitutions. For example, both heavy chains thatmake up the Fc region may comprise a single substitution, one chain maycomprise a single substitution and the other two substitutions, both cancontain two substitutions (although each chain will contain differentsubstitutions), etc.

In some embodiments, the CH2 and/or CH3 domain variants are made incombinations, that is, two or more variants per heavy chain Fc domain,selected from the group outlined above.

Other CH2 and/or CH3 domain variants that favor heterodimerization thatmay be employed in the design and preparation of the inventivemultispecific antibody analogs of the invention are provided in, forexample, Ridgeway et al., 1996, Protein Engineering 9[7]:617-621; U.S.Pat. No. 5,731,168; Xie et al., 2005, J Immunol Methods 296:95-101;Davis et al., 2010, Protein Engineering, Design & Selection23[4]:195-202; Gunasekaran et al., 2010, J Biol Chem 285[25]:1937-19646;and PCT/US2009/000071 (published as WO 2009/089004).

The Fc variants disclosed herein may be optimized for improved orreduced binding to Fc receptors or Fc ligands. By “Fc receptor” or “Fcligand” as used herein is meant a molecule, preferably a polypeptide,from any organism that binds to the Fc region of an antibody to form anFc-ligand complex. Fc ligands include but are not limited to FcγRs, (asdescribed above, including but not limited to FcγRIIIa, FcγRIIa,FcγRIIb, FcγRI and FcRn), C1q, C3, mannan binding lectin, mannosereceptor, staphylococcal protein A, streptococcal protein G, and viralFcγR. Fc ligands also include Fc receptor homologs (FcRH), which are afamily of Fc receptors that are homologous to the FcγRs. Fc ligands mayinclude undiscovered molecules that bind Fc.

The inventive multispecific antibody analogs may be designed to optimizeproperties, including but are not limited to enhanced or reducedaffinity for an Fc receptor. By “greater affinity” or “improvedaffinity” or “enhanced affinity” or “better affinity” than a parent Fcpolypeptide, as used herein, is meant that an Fc variant binds to an Fcreceptor with a significantly higher equilibrium constant of association(KA or Ka) or lower equilibrium constant of dissociation (KD or Kd) thanthe parent Fc polypeptide when the amounts of variant and parentpolypeptide in the binding assay are essentially the same. For example,the Fc variant with improved Fc receptor binding affinity may displayfrom about 5 fold to about 1000 fold, e.g. from about 10 fold to about500 fold improvement in Fc receptor binding affinity compared to theparent Fc polypeptide, where Fc receptor binding affinity is determined,for example, by the binding methods disclosed herein, including but notlimited to Biacore, by one skilled in the art. Accordingly, by “reducedaffinity” as compared to a parent Fc polypeptide as used herein is meantthat an Fc variant binds an Fc receptor with significantly lower KA orhigher KD than the parent Fc polypeptide. Greater or reduced affinitycan also be defined relative to an absolute level of affinity.

In one embodiment, particularly useful Fc modifications for the presentinvention are variants that reduce or ablate binding to one or moreFcγRs and/or complement proteins, thereby reducing or ablatingFc-mediated effector functions such as ADCC, ADCP, and CDC. Suchvariants are also referred to herein as “knockout variants” or “KOvariants”. Variants that reduce binding to FcγRs and complement areuseful for reducing unwanted interactions mediated by the Fc region andfor tuning the selectivity of the inventive multispecific antibodyanalogs. Preferred knockout variants are described in U.S. Ser. No.11/981,606, filed Oct. 31, 2007, entitled “Fc Variants with OptimizedProperties”. Preferred modifications include but are not limitedsubstitutions, insertions, and deletions at positions 234, 235, 236,237, 267, 269, 325, and 328, wherein numbering is according to the EUindex. Preferred substitutions include but are not limited to 234G,235G, 236R, 237K, 267R, 269R, 325L, and 328R, wherein numbering isaccording to the EU index. A preferred variant comprises 236R/328R.Variants may be used in the context of any IgG isotype or IgG isotype Fcregion, including but not limited to human IgG1, IgG2, IgG3, and/or IgG4and combinations thereof. Preferred IgG Fc regions for reducing FcγR andcomplement binding and reducing Fc-mediated effector functions are IgG2and IgG4 Fc regions. Hybrid isotypes may also be useful, for examplehybrid IgG1/IgG2 isotypes as described in US 2006-0134105. Othermodifications for reducing FcγR and complement interactions include butare not limited to substitutions 297A, 234A, 235A, 237A, 318A, 228P,236E, 268Q, 309L, 330S, 331S, 220S, 226S, 229S, 238S, 233P, and 234V, aswell as removal of the glycosylation at position 297 by mutational orenzymatic means or by production in organisms such as bacteria that donot glycosylate proteins. These and other modifications are reviewed inStrohl, 2009, Current Opinion in Biotechnology 20:685-691.

Fc modifications that improve binding to FcγRs and/or complement arealso amenable to incorporation in the design and preparation of theinventive multispecific antibody analogs disclosed herein. Such Fcvariants may enhance Fc-mediated effector functions such as ADCC, ADCP,and/or CDC. Preferred modifications for improving FcγR and complementbinding are described in, e.g., U.S. Pat. No. 8,188,231 and US2006-0235208. Preferred modifications comprise a substitution at aposition selected from the group consisting of 236, 239, 268, 324, and332, wherein numbering is according to the EU index. Preferredsubstitutions include but are not limited to 236A, 239D, 239E, 268D,267E, 268E, 268F, 324T, 332D, and 332E. Preferred variants include butare not limited to 239D/332E, 236A/332E, 236A/239D/332E, 268F/324T,267E/268F, 267E/324T, and 267E/268F/324T. Other modifications forenhancing FcγR and complement interactions include but are not limitedto substitutions 298A, 333A, 334A, 326A, 2471, 339D, 339Q, 280H, 290S,298D, 298V, 243L, 292P, 300L, 396L, 3051, and 396L. These and othermodifications are reviewed in Strohl, 2009, ibid.

In one embodiment, the inventive multispecific antibody analogsdisclosed herein may incorporate Fc variants that enhance affinity foran inhibitory receptor FcγRIIb. Such variants may provide the inventivemultispecific antibody analogs herein with immunomodulatory activitiesrelated to FcγRIIb+ cells, including for example B cells and monocytes.In one embodiment, the Fc variants provide selectively enhanced affinityto FcγRIIb relative to one or more activating receptors. Modificationsfor altering binding to FcγRIIb are described in U.S. Pat. No.8,063,187, filed May 30, 2008, entitled “Methods and Compositions forInhibiting CD32b Expressing Cells”. In particular, Fc variants thatimprove binding to FcγRIIb may include one or more modifications at aposition selected from the group consisting of 234, 235, 236, 237, 239,266, 267, 268, 325, 326, 327, 328, and 332, according to the EU index.Preferable substitutions for enhancing FcγRIIb affinity include but arenot limited to 234D, 234E, 234W, 235D, 235F, 235R, 235Y, 236D, 236N,237D, 237N, 239D, 239E, 266M, 267D, 267E, 268D, 268E, 327D, 327E, 328F,328W, 328Y, and 332E. More preferably, substitutions include but are notlimited to 235Y, 236D, 239D, 266M, 267E, 268D, 268E, 328F, 328W, and328Y. Preferred Fc variants for enhancing binding to FcγRIIb include butare not limited to 235Y/267E, 236D/267E, 239D/268D, 239D/267E,267E/268D, 267E/268E, and 267E/328F.

In some embodiments, the inventive multispecific antibody analogsdisclosed herein may incorporate Fc variants that improve FcRn binding.Such variants may enhance the in vivo pharmacokinetic properties of theinventive multispecific antibody analogs. Preferred variants thatincrease binding to FcRn and/or improve pharmacokinetic propertiesinclude but are not limited to substitutions at positions 259, 308, 428,and 434, including but not limited to for example 2591, 308F, 428L,428M, 434S, 434H, 434F, 434Y, 434M, 428L/4345, 2591/308F and2591/308F/428L (and others described in U.S. Ser. No. 12/341,769, filedDec. 22, 2008, entitled “Fc Variants with Altered Binding to FcRn”).Other variants that increase Fc binding to FcRn include but are notlimited to: 250E, 250Q, 428L, 428F, 250Q/428L (Hinton et al., 2004, J.Biol. Chem. 279(8): 6213-6216, Hinton et al. 2006 Journal of Immunology176:346-356), 256A, 272A, 286A, 305A, 307A, 307Q, 311A, 312A, 376A,378Q, 380A, 382A, 434A (Shields et al, Journal of Biological Chemistry,2001, 276(9):6591-6604), 252F, 252T, 252Y, 252W, 254T, 256S, 256R, 256Q,256E, 256D, 256T, 309P, 311S, 433R, 433S, 4331, 433P, 433Q, 434H, 434F,434Y, 252Y/254T/256E, 433K/434F/436H, 308T/309P/311S (Dall Acqua et al.Journal of Immunology, 2002, 169:5171-5180, Dall'Acqua et al., 2006,Journal of Biological Chemistry 281:23514-23524). Other modificationsfor modulating FcRn binding are described in Yeung et al., 2010, JImmunol, 182:7663-7671.

The inventive multispecific antibody analogs disclosed herein canincorporate Fc modifications in the context of any IgG isotype or IgGisotype Fc region, including but not limited to human IgG1, IgG2, IgG3,and/or IgG4. The IgG isotype may be selected such as to alter FcγR-and/or complement-mediated effector function(s). Hybrid IgG isotypes mayalso be useful. For example, US 2006-0134105 describes a number ofhybrid IgG1/IgG2 constant regions that may find use in the particularinvention. In some embodiments of the invention, inventive multispecificantibody analogs may comprise means for isotypic modifications, that is,modifications in a parent IgG to the amino acid type in an alternateIgG. For example, an IgG1/IgG3 hybrid variant may be constructed by asubstitutional means for substituting IgG1 positions in the CH2 and/orCH3 region with the amino acids from IgG3 at positions where the twoisotypes differ. Thus a hybrid variant IgG antibody may be constructedthat comprises one or more substitutional means, e.g., 274Q, 276K, 300F,339T, 356E, 358M, 384S, 392N, 397M, 4221, 435R, and 436F. In otherembodiments of the invention, an IgG1/IgG2 hybrid variant may beconstructed by a substitutional means for substituting IgG2 positions inthe CH2 and/or CH3 region with amino acids from IgG1 at positions wherethe two isotypes differ. Thus a hybrid variant IgG antibody may beconstructed that comprises one or more substitutional means, e.g., oneor more of the following amino acid substations: 233E, 234L, 235L, −236G(referring to an insertion of a glycine at position 236), and 327A.

All antibodies contain carbohydrate at conserved positions in theconstant regions of the heavy chain. Each antibody isotype has adistinct variety of N-linked carbohydrate structures. Aside from thecarbohydrate attached to the heavy chain, up to 30% of human IgGs have aglycosylated Fab region. IgG has a single N-linked biantennarycarbohydrate at Asn297 of the CH2 domain. For IgG from either serum orproduced ex vivo in hybridomas or engineered cells, the IgG areheterogeneous with respect to the Asn297 linked carbohydrate. For humanIgG, the core oligosaccharide normally consists of GlcNAc2Man3GlcNAc,with differing numbers of outer residues.

The inventive multispecific antibody analogs herein may also comprisecarbohydrate moieties, which moieties will be described with referenceto commonly used nomenclature for the description of oligosaccharides. Areview of carbohydrate chemistry which uses this nomenclature is foundin Hubbard et al. 1981, Ann. Rev. Biochem. 50:555-583. This nomenclatureincludes, for instance, Man, which represents mannose; GlcNAc, whichrepresents 2-N-acetylglucosamine; Gal which represents galactose; Fucfor fucose; and Glc, which represents glucose. Sialic acids aredescribed by the shorthand notation NeuNAc, for 5-N-acetylneuraminicacid, and NeuNGc for 5-glycolylneuraminic.

The term “glycosylation” means the attachment of oligosaccharides(carbohydrates containing two or more simple sugars linked together e.g.from two to about twelve simple sugars linked together) to aglycoprotein. The oligosaccharide side chains are typically linked tothe backbone of the glycoprotein through either N- or 0-linkages. Theoligosaccharides of inventive multispecific antibody analogs disclosedherein occur generally are attached to a CH2 domain of an Fc region asN-linked oligosaccharides. “N-linked glycosylation” refers to theattachment of the carbohydrate moiety to an asparagine residue in aglycoprotein chain. The skilled artisan will recognize that, forexample, each of murine IgG1, IgG2a, IgG2b and IgG3 as well as humanIgG1, IgG2, IgG3, IgG4, IgA and IgD CH2 domains have a single site forN-linked glycosylation at residue 297.

For the purposes herein, a “mature core carbohydrate structure” refersto a processed core carbohydrate structure attached to an Fc regionwhich generally consists of the following carbohydrate structureGlcNAc(Fucose)-GlcNAc-Man-(Man-GlcNAc)2 typical of biantennaryoligosaccharides. The mature core carbohydrate structure is attached tothe Fc region of the glycoprotein, generally via N-linkage to Asn297 ofa CH2 domain of the Fc region. A “bisecting GlcNAc” is a GlcNAc residueattached to the α1,4 mannose of the mature core carbohydrate structure.The bisecting GlcNAc can be enzymatically attached to the mature corecarbohydrate structure by a α(1,4)-N-acetylglucosaminyltransferase IIIenzyme (GnTIII). CHO cells do not normally express GnTIII (Stanley etal., 1984, J. Biol. Chem. 261:13370-13378), but may be engineered to doso (Umana et al., 1999, Nature Biotech. 17:176-180).

Described herein are multispecific antibody analogs that comprisemodified glycoforms or engineered glycoforms. By “modified glycoform” or“engineered glycoform” as used herein is meant a carbohydratecomposition that is covalently attached to a protein, for example anantibody, wherein said carbohydrate composition differs chemically fromthat of a parent protein. Engineered glycoforms may be useful for avariety of purposes, including but not limited to enhancing or reducingFcγR-mediated effector function. In one embodiment, the inventivemultispecific antibody analogs disclosed herein are modified to controlthe level of fucosylated and/or bisecting oligosaccharides that arecovalently attached to the Fc region.

A variety of methods are well known in the art for generating modifiedglycoforms (Umana et al., 1999, Nat Biotechnol 17:176-180; Davies etal., 2001, Biotechnol Bioeng 74:288-294; Shields et al., 2002, J BiolChem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473;U.S. Ser. No. 12/434,533). These techniques control the level offucosylated and/or bisecting oligosaccharides that are covalentlyattached to the Fc region, for example by expressing an IgG in variousorganisms or cell lines, engineered or otherwise (for example Lec-13 CHOcells or rat hybridoma YB2/0 cells), by regulating enzymes involved inthe glycosylation pathway (for example FUT8 [α-1,6-fucosyltranserase]and/or β1-4-N-acetylglucosaminyltransferase III [GnTIII]), by modifyingcarbohydrate(s) after the IgG has been expressed, or by expressingantibody in the presence of fucose analogs as enzymatic inhibitors.Other methods for modifying glycoforms of the inventive multispecificantibody analogs disclosed herein include using glycoengineered strainsof yeast (Li et al., 2006, Nature Biotechnology 24(2):210-215), moss(Nechansky et al., 2007, Mol Immunol 44(7):1826-8), and plants (Cox etal., 2006, Nat Biotechnol 24(12):1591-7). The use of a particular methodto generate a modified glycoform is not meant to constrain embodimentsto that method. Rather, embodiments disclosed herein encompass inventivemultispecific antibody analogs with modified glycoforms irrespective ofhow they are produced.

In one embodiment, the inventive multispecific antibody analogsdisclosed herein are glycoengineered to alter the level of sialylation.Higher levels of sialylated Fc glycans in immunoglobulin G molecules canadversely impact functionality (Scallon et al., 2007, Mol. Immunol.44(7):1524-34), and differences in levels of Fc sialylation can resultin modified anti-inflammatory activity (Kaneko et al., 2006, Science313:670-673). Because antibodies may acquire anti-inflammatoryproperties upon sialylation of Fc core polysaccharide, it may beadvantageous to glycoengineer the inventive multispecific antibodyanalogs disclosed herein for greater or reduced Fc sialic acid content.

“Engineered glycoform” typically refers to the different carbohydrate oroligosaccharide; thus for example an immunoglobulin may comprise anengineered glycoform. In one embodiment, a composition disclosed hereincomprises a glycosylated inventive multispecific antibody analog havingan Fc region, wherein about 51-100% of the glycosylated antibody, e.g.,80-100%, 90-100%, 95-100%, etc. of the antibody in the compositioncomprises a mature core carbohydrate structure which lacks fucose. Inanother embodiment, the antibody in the composition both comprises amature core carbohydrate structure that lacks fucose and additionallycomprises at least one amino acid modification in the Fc region. In analternative embodiment, a composition comprises a glycosylated inventivemultispecific antibody analog having an Fc region, wherein about 51-100%of the glycosylated antibody, 80-100%, or 90-100%, of the antibody inthe composition comprises a mature core carbohydrate structure whichlacks sialic acid. In another embodiment, the antibody in thecomposition both comprises a mature core carbohydrate structure thatlacks sialic acid and additionally comprises at least one amino acidmodification in the Fc region. In yet another embodiment, a compositioncomprises a glycosylated inventive multispecific antibody analog havingan Fc region, wherein about 51-100% of the glycosylated antibody,80-100%, or 90-100%, of the antibody in the composition comprises amature core carbohydrate structure which contains sialic acid. Inanother embodiment, the antibody in the composition both comprises amature core carbohydrate structure that contains sialic acid andadditionally comprises at least one amino acid modification in the Fcregion. In another embodiment, the combination of engineered glycoformand amino acid modification provides optimal Fc receptor bindingproperties to the antibody.

The inventive multispecific antibody analogs disclosed herein maycomprise one or more modifications that provide additional optimizedproperties. Said modifications may be amino acid modifications, or maybe modifications that are made enzymatically or chemically. Suchmodification(s) likely provide some improvement in the inventivemultispecific antibody analog, for example an enhancement in itsstability, solubility, function, or clinical use. Disclosed herein are avariety of improvements that may be made by coupling the inventivemultispecific antibody analogs disclosed herein with additionalmodifications.

In one embodiment, at least one variable region of multispecificantibody analog disclosed herein may be affinity matured, that is to saythat amino acid modifications have been made in the VH and/or VL domainsto enhance binding of the antibody to its target antigen. Such types ofmodifications may improve the association and/or the dissociationkinetics for binding to the target antigen. Other modifications includethose that improve selectivity for target antigen vs. alternativetargets. These include modifications that improve selectivity forantigen expressed on target vs. non-target cells. Inventivemultispecific antibody analogs disclosed herein may comprise one or moremodifications that provide reduced or enhanced internalization of aninventive multispecific antibody analog.

In other embodiments, modifications are made to improve biophysicalproperties of the inventive multispecific antibody analogs disclosedherein, including but not limited to stability, solubility, andoligomeric state. Modifications can include, for example, substitutionsthat provide more favorable intramolecular interactions in the inventivemultispecific antibody analog such as to provide greater stability, orsubstitution of exposed nonpolar amino acids with polar amino acids forhigher solubility. Other modifications to the inventive multispecificantibody analogs disclosed herein include those that enable the specificformation or homodimeric or homomultimeric molecules. Such modificationsinclude but are not limited to engineered disulfides, as well aschemical modifications or aggregation methods.

In further embodiments, the inventive multispecific antibody analogsdisclosed herein comprise modifications that remove proteolyticdegradation sites. These may include, for example, protease sites thatreduce production yields, as well as protease sites that degrade theadministered protein in vivo. In one embodiment, additionalmodifications are made to remove covalent degradation sites such asdeamidation (i.e. deamidation of glutaminyl and asparaginyl residues tothe corresponding glutamyl and aspartyl residues), oxidation, andproteolytic degradation sites. Deamidation sites that are particularuseful to remove are those that have enhance propensity for deamidation,including, but not limited to asparaginyl and gltuamyl residues followedby glycines (NG and QG motifs, respectively). In such cases,substitution of either residue can significantly reduce the tendency fordeamidation. Common oxidation sites include methionine and cysteineresidues. Other covalent modifications, that can either be introduced orremoved, include hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of the“-amino groups of lysine, arginine, and histidine side chains,acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group. Additional modifications also may include but are notlimited to posttranslational modifications such as N-linked or O-linkedglycosylation and phosphorylation.

Modifications may include those that improve expression and/orpurification yields from hosts or host cells commonly used forproduction of biologics. These include, but are not limited to variousmammalian cell lines (e.g. CHO, HEK, COS, NIH LT3, Saos, and the like),yeast cells, bacterial cells, and plant cells. Additional modificationsinclude modifications that remove or reduce the ability of heavy chainsto form inter-chain disulfide linkages. Additional modifications includemodifications that remove or reduce the ability of heavy chains to formintra-chain disulfide linkages.

The inventive multispecific antibody analogs disclosed herein maycomprise modifications that include the use of unnatural amino acidsincorporated using, including but not limited to methods described inLiu & Schultz, 2010, Annu Rev Biochem 79:413-444. In some embodiments,these modifications enable manipulation of various functional,biophysical, immunological, or manufacturing properties discussed above.In additional embodiments, these modifications enable additionalchemical modification for other purposes.

Other modifications are contemplated herein. For example, the inventivemultispecific antibody analogs may be linked to one of a variety ofnonproteinaceous polymers, e.g., polyethylene glycol (PEG),polypropylene glycol, polyoxyalkylenes, or copolymers of polyethyleneglycol and polypropylene glycol. Additional amino acid modifications maybe made to enable specific or non-specific chemical or posttranslationalmodification of the inventive multispecific antibody analogs. Suchmodifications, include, but are not limited to PEGylation andglycosylation. Specific substitutions that can be utilized to enablePEGylation include, but are not limited to, introduction of novelcysteine residues or unnatural amino acids such that efficient andspecific coupling chemistries can be used to attach a PEG or otherwisepolymeric moiety. Introduction of specific glycosylation sites can beachieved by introducing novel N-X-T/S sequences into the inventivemultispecific antibody analogs disclosed herein.

Modifications to reduce immunogenicity may include modifications thatreduce binding of processed peptides derived from the parent sequence toMHC proteins. For example, amino acid modifications would be engineeredsuch that there are no or a minimal number of immune epitopes that arepredicted to bind, with high affinity, to any prevalent MHC alleles.Several methods of identifying MHC-binding epitopes in protein sequencesare known in the art and may be used to score epitopes in an antibodydisclosed herein.

Covalent modifications are included within the scope of inventivemultispecific antibody analogs disclosed herein, and are generally, butnot always, done post-translationally. For example, several types ofcovalent modifications can be introduced into the molecule by reactingspecific amino acid residues with an organic derivatizing agent that iscapable of reacting with selected side chains or the N- or C-terminalresidues. In some embodiments, the covalent modification of theinventive multispecific antibody analogs disclosed herein comprises theaddition of one or more labels. The term “labeling group” means anydetectable label. In some embodiments, the labeling group is coupled tothe inventive multispecific antibody analog via spacer arms of variouslengths to reduce potential steric hindrance. Various methods forlabeling proteins are known in the art and may be used in generatinginventive multispecific antibody analogs disclosed herein.

In certain embodiments, the inventive multispecific antibody analogsdisclosed herein comprise “fusion proteins”, also referred to herein as“conjugates”. The fusion partner or conjugate partner can beproteinaceous or non-proteinaceous; the latter generally being generatedusing functional groups on the inventive multispecific antibody analogand on the conjugate partner. Conjugate and fusion partners may be anymolecule, including small molecule chemical compounds and polypeptides.For example, a variety of conjugates and methods are described in Trailet al., 1999, Curr. Opin. Immunol. 11:584-588. Possible conjugatepartners include but are not limited to cytokines, cytotoxic agents,toxins, radioisotopes, chemotherapeutic agent, anti-angiogenic agents, atyrosine kinase inhibitors, and other therapeutically active agents. Insome embodiments, conjugate partners may be thought of more as payloads,that is to say that the goal of a conjugate is targeted delivery of theconjugate partner to a targeted cell, for example a cancer cell orimmune cell, by the multispecific antibody analogs. Thus, for example,the conjugation of a toxin to a multispecific antibody analog targetsthe delivery of said toxin to cells expressing the target antigen. Aswill be appreciated by one skilled in the art, in reality the conceptsand definitions of fusion and conjugate are overlapping. The designationof a fusion or conjugate is not meant to constrain it to any particularembodiment disclosed herein. Rather, these terms are used to convey thebroad concept that any multispecific antibody analogs disclosed hereinmay be linked genetically, chemically, or otherwise, to one or morepolypeptides or molecules to provide some desirable property.

Suitable conjugates include, but are not limited to, labels as describedbelow, drugs and cytotoxic agents including, but not limited to,cytotoxic drugs (e.g., chemotherapeutic agents) or toxins or activefragments of such toxins. Suitable toxins and their correspondingfragments include diphtheria A chain, exotoxin A chain, ricin A chain,abrin A chain, curcin, crotin, phenomycin, enomycin and the like.Cytotoxic agents also include radiochemicals made by conjugatingradioisotopes to inventive multispecific antibody analog, or binding ofa radionuclide to a chelating agent that has been covalently attached tothe inventive multispecific antibody analog. Additional embodimentsutilize calicheamicin, auristatins, geldanamycin, maytansine, andduocarmycins and analogs. Antibody-drug conjugates are described inAlley et al., 2010, Curr Opin Chem Biol 14[4]:529-37.

In certain embodiments, the inventive multispecific antibody analogsdisclosed herein are fused or conjugated to a cytokine By “cytokine” asused herein is meant a generic term for proteins released by one cellpopulation that act on another cell as intercellular mediators. Forexample, as described in Penichet et al., 2001, J. Immunol. Methods248:91-101, cytokines may be fused to an inventive multispecificantibody analog to provide an array of desirable properties. Examples ofsuch cytokines are lymphokines, monokines, and traditional polypeptidehormones. Included among the cytokines are growth hormone such as humangrowth hormone, N-methionyl human growth hormone, and bovine growthhormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin;prorelaxin; glycoprotein hormones such as follicle stimulating hormone(FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH);hepatic growth factor; fibroblast growth factor; prolactin; placentallactogen; tumor necrosis factor-alpha and -beta; mullerian-inhibitingsubstance; mouse gonadotropin-associated peptide; inhibin; activin;vascular endothelial growth factor; integrin; thrombopoietin (TPO);nerve growth factors such as NGF-beta; platelet-growth factor;transforming growth factors (TGFs) such as TGF-alpha and TGF-beta;insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon-alpha, beta, and-gamma; colony stimulating factors (CSFs) such as macrophage-CSF(M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF(G-CSF); interleukins (ILs) such as IL-1, IL-1alpha, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, a tumornecrosis factor such as TNF-alpha or TNF-beta; C5a; and otherpolypeptide factors including LIF and kit ligand (KL). As used herein,the term cytokine includes proteins from natural sources or fromrecombinant cell culture, and biologically active equivalents of thenative sequence cytokines.

In further embodiments, the inventive multispecific antibody analogsdisclosed herein may be conjugated to a “receptor” (such streptavidin)for utilization in tumor pretargeting wherein the analog-receptorconjugate is administered to the patient, followed by removal of unboundconjugate from the circulation using a clearing agent and thenadministration of a “ligand” (e.g. avidin) which is conjugated to acytotoxic agent (e.g. a radionucleotide). In an alternate embodiment,the inventive multispecific antibody analog is conjugated or operablylinked to an enzyme in order to employ Antibody Dependent EnzymeMediated Prodrug Therapy (ADEPT). ADEPT may be used by conjugating oroperably linking the inventive multispecific antibody analog to aprodrug-activating enzyme that converts a prodrug (e.g. a peptidylchemotherapeutic agent.

Also disclosed herein are methods for producing and experimentallytesting the inventive multispecific antibody analogs. The disclosedmethods are not meant to constrain embodiments to any particularapplication or theory of operation. Rather, the provided methods aremeant to illustrate generally that one or more multispecific antibodyanalogs of the invention may be produced and experimentally tested toobtain inventive multispecific antibody analogs. General methods forantibody molecular biology, expression, purification, and screening aredescribed in Antibody Engineering, edited by Kontermann & Dubel,Springer, Heidelberg, 2001; and Hayhurst & Georgiou, 2001, Curr OpinChem Biol 5:683-689; Maynard & Georgiou, 2000, Annu Rev Biomed Eng2:339-76.

In one embodiment disclosed herein, nucleic acids are created thatencode the inventive multispecific antibody analogs, and that may thenbe cloned into host cells, such as yeast cells or mammalian cells,expressed and assayed, if desired. Thus, nucleic acids, and particularlyDNA, may be made that encode each protein sequence. These practices arecarried out using well-known procedures. For example, a variety ofmethods that may find use in generating inventive multispecific antibodyanalogs disclosed herein are described in Molecular Cloning—A LaboratoryManual, 3rd Ed. (Maniatis, Cold Spring Harbor Laboratory Press, NewYork, 2001), and Current Protocols in Molecular Biology (John Wiley &Sons). There are a variety of techniques that may be used to efficientlygenerate DNA encoding inventive multispecific antibody analogs disclosedherein. Such methods include but are not limited to gene assemblymethods, PCR-based method and methods which use variations of PCR,ligase chain reaction-based methods, pooled oligo methods such as thoseused in synthetic shuffling, error-prone amplification methods andmethods which use oligos with random mutations, classical site-directedmutagenesis methods, cassette mutagenesis, and other amplification andgene synthesis methods. As is known in the art, there are a variety ofcommercially available kits and methods for gene assembly, mutagenesis,vector subcloning, and the like, and such commercial products find usein for generating nucleic acids that encode inventive multispecificantibody analogs.

The inventive multispecific antibody analogs disclosed herein may beproduced by culturing a host cell transformed with nucleic acid, e.g.,expression vectors containing nucleic acid encoding the first and secondpolypeptides of inventive multispecific antibody analogs, under theappropriate conditions to induce or cause expression of thepolypeptides. The conditions appropriate for expression will vary withthe choice of the expression vector and the host cell, and will beeasily ascertained by one skilled in the art through routineexperimentation. A wide variety of appropriate host cells may be used,including but not limited to mammalian cells, bacteria, insect cells,yeast cells, and plant cells. For example, a variety of cell lines thatmay find use in generating inventive multispecific antibody analogsdisclosed herein are described in the ATCC® cell line catalog, availablefrom the American Type Culture Collection.

In certain embodiments, the inventive multispecific antibody analogs areexpressed in mammalian expression systems, including systems in whichthe expression constructs are introduced into the mammalian cells usingvirus such as retrovirus or adenovirus. Any mammalian cells may be used,e.g., human, mouse, rat, hamster, and primate cells. Suitable cells alsoinclude known research cells, including but not limited to Jurkat Tcells, NIH3T3, CHO, BHK, COS, HEK293, PER C.6, HeLa, Sp2/0, NS0 cellsand variants thereof. In an alternate embodiment, library proteins areexpressed in bacterial cells. Bacterial expression systems are wellknown in the art, and include Escherichia coli (E. coli), Bacillussubtilis, Streptococcus cremoris, and Streptococcus lividans. Inalternate embodiments, inventive multispecific antibody analogs areproduced in insect cells (e.g. Sf21/Sf9, Trichoplusia ni Bti-Tn5b1-4) oryeast cells (e.g. S. cerevisiae, Pichia, etc.). In an alternateembodiment, inventive multispecific antibody analogs are expressed invitro using cell free translation systems. In vitro translation systemsderived from both prokaryotic (e.g. E. coli) and eukaryotic (e.g. wheatgerm, rabbit reticulocytes) cells are available and may be chosen basedon the expression levels and functional properties of the protein ofinterest. For example, as appreciated by those skilled in the art, invitro translation is required for some display technologies, for exampleribosome display. In addition, the inventive multispecific antibodyanalogs may be produced by chemical synthesis methods. Also transgenicexpression systems both animal (e.g. cow, sheep or goat milk,embryonated hen's eggs, whole insect larvae, etc.) and plant (e.g. corn,tobacco, duckweed, etc.)

The nucleic acids that encode the first and second polypeptides ofinventive multispecific antibody analogs disclosed herein may beincorporated into one or more expression vectors, as appropriate, inorder to express the encoded polypeptides. A variety of expressionvectors may be utilized for protein expression. Expression vectors maycomprise self-replicating extra-chromosomal vectors or vectors whichintegrate into a host genome. Expression vectors are constructed to becompatible with the host cell type. Thus expression vectors which finduse in generating inventive multispecific antibody analogs disclosedherein include but are not limited to those which enable proteinexpression in mammalian cells, bacteria, insect cells, yeast cells, andin vitro systems. As is known in the art, a variety of expressionvectors are available, commercially or otherwise, that may find use forexpressing inventive multispecific antibody analogs disclosed herein.

Expression vectors typically comprise a protein or polypeptide to beexpressed, which is operably linked with control or regulatorysequences, selectable markers, any fusion partners, and/or additionalelements. By “operably linked” herein is meant that the nucleic acid isplaced into a functional relationship with another nucleic acidsequence. Generally, these expression vectors include transcriptionaland translational regulatory nucleic acid operably linked to the nucleicacid encoding the inventive multispecific antibody analog, and aretypically appropriate to the host cell used to express the protein. Ingeneral, the transcriptional and translational regulatory sequences mayinclude promoter sequences, ribosomal binding sites, transcriptionalstart and stop sequences, translational start and stop sequences, andenhancer or activator sequences. As is also known in the art, expressionvectors typically contain a selection gene or marker to allow theselection of transformed host cells containing the expression vector.Selection genes are well known in the art and will vary with the hostcell used.

The first and second polypeptides of the invention may each beindependently operably linked to a fusion partner to enable targeting ofthe expressed polypeptide and/or multispecific antibody analog,purification, screening, display, and the like. Fusion partners may belinked to the inventive multispecific antibody analog sequence via alinker sequences. The linker sequence will generally comprise a smallnumber of amino acids, typically less than ten, although longer linkersmay also be used. Typically, linker sequences are selected to beflexible and resistant to degradation. As will be appreciated by thoseskilled in the art, any of a wide variety of sequences may be used aslinkers. For example, a common linker sequence comprises the amino acidsequence GGGGS (SEQ ID NO: 94). A fusion partner may be a targeting orsignal sequence that directs inventive multispecific antibody analog andany associated fusion partners to a desired cellular location or to theextracellular media. As is known in the art, certain signaling sequencesmay target a protein to be either secreted into the growth media, orinto the periplasmic space, located between the inner and outer membraneof the cell. A fusion partner may also be a sequence that encodes apeptide or protein that enables purification and/or screening. Suchfusion partners include but are not limited to polyhistidine tags(His-tags) (for example H₆ (SEQ ID NO: 95) and H₁₀ (SEQ ID NO: 96) orother tags for use with Immobilized Metal Affinity Chromatography (IMAC)systems (e.g. Ni+2 affinity columns)), GST fusions, MBP fusions,Strep-tag, the BSP biotinylation target sequence of the bacterial enzymeBirA, and epitope tags which are targeted by antibodies (for examplec-myc tags, flag-tags, and the like). As will be appreciated by thoseskilled in the art, such tags may be useful for purification, forscreening, or both. For example, an inventive multispecific antibodyanalog may be purified using a His-tag by immobilizing it to a Ni+2affinity column, and then after purification the same His-tag may beused to immobilize the antibody to a Ni+2 coated plate to perform anELISA or other binding assay (as described below). A fusion partner mayenable the use of a selection method to screen inventive multispecificantibody analogs (see below). Fusion partners that enable a variety ofselection methods are well-known in the art.

For example, by fusing the members of an inventive multispecificantibody analog library to the gene III protein, phage display can beemployed. Fusion partners may enable inventive multispecific antibodyanalogs to be labeled. Alternatively, a fusion partner may bind to aspecific sequence on the expression vector, enabling the fusion partnerand associated inventive multispecific antibody analog to be linkedcovalently or noncovalently with the nucleic acid that encodes them. Themethods of introducing exogenous nucleic acid into host cells are wellknown in the art, and will vary with the host cell used. Techniquesinclude but are not limited to dextran-mediated transfection, calciumphosphate precipitation, calcium chloride treatment, polybrene mediatedtransfection, protoplast fusion, electroporation, viral or phageinfection, encapsulation of the polynucleotide(s) in liposomes, anddirect microinjection of the DNA into nuclei. In the case of mammaliancells, transfection may be either transient or stable.

In certain embodiments, the multispecific antibody analogs are purifiedor isolated after expression. The multispecific antibody analogs may beisolated or purified in a variety of ways known to those skilled in theart. Purification may be particularly useful in the invention forseparating heterodimeric heavy chain species from homodimeric heavychain species, as described herein. Standard purification methodsinclude chromatographic techniques, including ion exchange, hydrophobicinteraction, affinity, sizing or gel filtration, and reversed-phase,carried out at atmospheric pressure or at high pressure using systemssuch as FPLC and HPLC. Purification methods also includeelectrophoretic, isoelectric focusing, immunological, precipitation,dialysis, and chromatofocusing techniques. Ultrafiltration anddiafiltration techniques, in conjunction with protein concentration, arealso useful. As is well known in the art, a variety of natural proteinsbind Fc and antibodies, and these proteins can find use for purificationof inventive multispecific antibody analogs disclosed herein. Forexample, the bacterial proteins A and G bind to the Fc region. Likewise,the bacterial protein L binds to the Fab region of some antibodies, asof course does the antibody's target antigen. Purification can often beenabled by a particular fusion partner. For example, inventivemultispecific antibody analogs may be purified using glutathione resinif a GST fusion is employed, Ni+2 affinity chromatography if a His-tagis employed, or immobilized anti-flag antibody if a flag-tag is used.For general guidance in suitable purification techniques, see, e.g.Protein Purification: Principles and Practice, 3rd Ed., Scopes,Springer-Verlag, NY, 1994. The degree of purification necessary willvary depending on the screen or use of the inventive multispecificantibody analogs. In some instances no purification is necessary. Forexample in one embodiment, if the inventive multispecific antibodyanalogs are secreted, screening may take place directly from the media.As is well known in the art, some methods of selection do not involvepurification of proteins.

Virtually any antigen may be targeted by the inventive multispecificantibody analogs disclosed herein, including but not limited toproteins, subunits, domains, motifs, and/or epitopes belonging to thefollowing list of target antigens, which includes both soluble factorssuch as cytokines and membrane-bound factors, including transmembranereceptors: 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1Adenosine Receptor, A33, ACE, ACE-2, Activin, Activin A, Activin AB,Activin B, Activin C, Activin RIA, Activin RIA ALK-2, Activin RIB ALK-4,Activin RIIA, Activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17/TACE,ADAMS, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, Addressins, aFGF, ALCAM, ALK,ALK-1, ALK-7, alpha-1-antitrypsin, alpha-V/beta-1 antagonist, ANG, Ang,APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, Artemin, anti-Id, ASPARTIC,Atrial natriuretic factor, av/b3 integrin, Axl, b2M, B7-1, B7-2, B7-H,B-lymphocyte Stimulator (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1,BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM,BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3 Osteogenin, BMP-4 BMP-2b,BMP-5, BMP-6 Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA(ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMPs, b-NGF,BOK, Bombesin, Bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC,complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8,Calcitonin, cAMP, carcinoembryonic antigen (CEA), carcinoma-associatedantigen, Cathepsin A, Cathepsin B, Cathepsin C/DPPI, Cathepsin D,Cathepsin E, Cathepsin H, Cathepsin L, Cathepsin O, Cathepsin S,Cathepsin V, Cathepsin X/Z/P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12,CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21,CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6,CCL7, CCL8, CCL9/10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5,CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8,CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20,CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67proteins), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54,CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123,CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR,cGMP, CINC, Clostridium botulinum toxin, Clostridium perfringens toxin,CKb8-1, CLC, CMV, CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK,CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5,CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14,CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6,cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, Decayaccelerating factor, des(1-3)-IGF-1 (brain IGF-1), Dhh, digoxin, DNAM-1,Dnase, Dpp, DPPIV/CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR(ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, Enkephalinase, eNOS,Eot, eotaxin1, EpCAM, Ephrin B2/EphB4, EPO, ERCC, E-selectin, ET-1,Factor IIa, Factor VII, Factor VIIIc, Factor IX, fibroblast activationprotein (FAP), Fas, FcR1, FEN-1, Ferritin, FGF, FGF-19, FGF-2, FGF3,FGF-8, FGFR, FGFR-3, Fibrin, FL, FLIP, Flt-3, Flt-4, Folliclestimulating hormone, Fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6,FZD7, FZD8, FZD9, FZD10, G250, Gas 6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1,GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7(BMP-12, CDMP-3), GDF-8 (Myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF,GFAP, GFRa-1, GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, Glucagon, Glut4, glycoprotein IIb/IIIa (GP IIb/IIIa), GM-CSF, gp130, gp72, GRO, Growthhormone releasing factor, Hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMVgB envelope glycoprotein, HCMV) gH envelope glycoprotein, HCMV UL,Hemopoietic growth factor (HGF), Hep B gp120, heparanase, Her2, Her2/neu(ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gBglycoprotein, HSV gD glycoprotein, HGFA, High molecular weightmelanoma-associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp120 V3loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin,human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, 1-309,IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF,IGF binding proteins, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R,IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10,IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon (INF)-alpha,INF-beta, INF-gamma, Inhibin, iNOS, Insulin A-chain, Insulin B-chain,Insulin-like growth factor 1, integrin alpha2, integrin alpha3, integrinalpha4, integrin alpha4/beta1, integrin alpha4/beta7, integrin alpha5(alphaV), integrin alpha5/beta1, integrin alpha5/beta3, integrin alpha6,integrin beta1, integrin beta2, interferon gamma, IP-10, I-TAC, JE,Kallikrein 2, Kallikrein 5, Kallikrein 6, Kallikrein 11, Kallikrein 12,Kallikrein 14, Kallikrein 15, Kallikrein L1, Kallikrein L2, KallikreinL3, Kallikrein L4, KC, KDR, Keratinocyte Growth Factor (KGF), laminin 5,LAMP, LAP, LAP (TGF-1), Latent TGF-1, Latent TGF-1 bp1, LBP, LDGF,LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3,Lfo, LIF, LIGHT, lipoproteins, LIX, LKN, Lptn, L-Selectin, LT-a, LT-b,LTB4, LTBP-1, Lung surfactant, Luteinizing hormone, Lymphotoxin BetaReceptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF,MDC, Mer, METALLOPROTEASES, MGDF receptor, MGMT, MHC(HLA-DR), MIF, MIG,MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13,MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo,MSK, MSP, mucin (Muc1), MUC18, Muellerian-inhibitin substance, Mug,MuSK, NAIP, NAP, NCAD, N-Cadherin, NCA 90, NCAM, NCAM, Neprilysin,Neurotrophin-3, -4, or -6, Neurturin, Neuronal growth factor (NGF),NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN,OSM, OX40L, OX40R, p150, p95, PADPr, Parathyroid hormone, PARC, PARP,PBR, PBSF, PCAD, P-Cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4,PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (FLAP),P1GF, PLP, PP14, Proinsulin, Prorelaxin, Protein C, PS, PSA, PSCA,prostate specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51,RANK, RANKL, RANTES, RANTES, Relaxin A-chain, Relaxin B-chain, renin,respiratory syncytial virus (RSV) F, RSV Fgp, Ret, Rheumatoid factors,RLIP76, RPA2, RSK, S100, SCF/KL, SDF-1, SERINE, Serum albumin, sFRP-3,Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat,STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72),TARC, TCA-3, T-cell receptors (e.g., T-cell receptor alpha/beta), TdT,TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular FLAP-like alkalinephosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan Specific,TGF-beta RI (ALK-5), TGF-beta RII, TGF-beta RII, TGF-beta Rill,TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, TGF-beta5, Thrombin, ThymusCk-1, Thyroid stimulating hormone, Tie, TIMP, TIQ, Tissue Factor,TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alpha beta, TNF-beta2, TNFc,TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1Apo-2, DR4), TNFRSF10B (TRAIL R2DRS,KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3DcR1, LIT, TRID),TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R),TNFRSF11B (OPG OCIF, TR1), TNFRSF12 (TWEAK R FN14), TNFRSF13B (TACI),TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16(NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR AITR), TNFRSF19 (TROYTAJ, TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF RI CD120a, p55-60),TNFRSF1B (TNF RII CD120b, p75-80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNFRIII, TNFC R), TNFRSF4 (OX40 ACT35, TXGP1 R), TNFRSF5 (CD40 p50),TNFRSF6 (Fas Apo-1, APT1, CD95), TNFRSF6B (DcR3M68, TR6), TNFRSF7(CD27), TNFRSF8 (CD30), TNFRSF9 (4-1BB CD137, ILA), TNFRSF21 (DR6),TNFRSF22 (DcTRAIL R2TNFRH2), TNFRST23 (DcTRAIL R1TNFRH1), TNFRSF25 (DR3Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 Ligand, TL2),TNFSF11 (TRANCE/RANK Ligand ODF, OPG Ligand), TNFSF12 (TWEAK Apo-3Ligand, DR3 Ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1,THANK, TNFSF20), TNFSF14 (LIGHT HVEM Ligand, LTg), TNFSF15 (TL1A/VEGI),TNFSF18 (GITR Ligand AITR Ligand, TL6), TNFSF1A (TNF-α Conectin, DIF,TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4(OX40 Ligand gp34, TXGP1), TNFSF5 (CD40 Ligand CD154, gp39, HIGM1, IMD3,TRAP), TNFSF6 (Fas Ligand Apo-1 Ligand, APT1 Ligand), TNFSF7 (CD27Ligand CD70), TNFSF8 (CD30 Ligand CD153), TNFSF9 (4-1BB Ligand CD137Ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE,transferring receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associatedantigen CA 125, tumor-associated antigen expressing Lewis Y relatedcarbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, Urokinase, VCAM, VCAM-1,VECAD, VE-Cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3(flt-4), VEGI, VIM, Viral antigens, VLA, VLA-1, VLA-4, VNR integrin, vonWillebrands factor, WIF-1, WNT1, WNT2, WNT2B/13, WNT3, WNT3A, WNT4,WNTSA, WNTSB, WNT6, WNTSA, WNTSB, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B,WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD,and receptors for hormones and growth factors.

Exemplary antigens that may be targeted specifically by themultispecific antibody analogs of the invention include but are notlimited to: CD20, CD19, Her2, EGFR, EpCAM, c-MET, CD3, FcγRIIIa (CD16),FcγRIIa (CD32a), FcγRIIb (CD32b), FcγRI (CD64), Toll-like receptors(TLRs) such as TLR4 and TLR9, cytokines such as IL-2, IL-5, IL-13,IL-12, IL-23, and TNFα, cytokine receptors such as IL-2R, chemokines,chemokine receptors, growth factors such as VEGF and HGF, and the like.

The choice of suitable target antigens and co-targets depends on thedesired therapeutic application. Some targets that have provenespecially amenable to antibody therapy are those with signalingfunctions. Other therapeutic antibodies exert their effects by blockingsignaling of the receptor by inhibiting the binding between a receptorand its cognate ligand. Another mechanism of action of therapeuticantibodies is to cause receptor down regulation. Other antibodies do notwork by signaling through their target antigen. The choice of co-targetswill depend on the detailed biology underlying the pathology of theindication that is being treated.

Monoclonal antibody therapy has emerged as an important therapeuticmodality for cancer (Weiner et al., 2010, Nature Reviews Immunology10:317-327; Reichert et al., 2005, Nature Biotechnology23[9]:1073-1078). For anti-cancer treatment it may be desirable totarget one antigen (antigen-1) whose expression is restricted to thecancerous cells while co-targeting a second antigen (antigen-2) thatmediates some immunological killing activity. For other treatments itmay be beneficial to co-target two antigens, for example two angiogenicfactors or two growth factors that are each known to play some role inproliferation of the tumor. Exemplary co-targets for oncology includebut are not limited to HGF and VEGF, IGF-1R and VEGF, Her2 and VEGF,CD19 and CD3, CD20 and CD3, Her2 and CD3, CD19 and FcγRIIIa, CD20 andFcγRIIIa, Her2 and FcγRIIIa. An inventive multispecific antibody analogof the invention may be capable of binding VEGF and phosphatidylserine;VEGF and ErbB3; VEGF and PLGF; VEGF and ROBO4; VEGF and BSG2; VEGF andCDCP1; VEGF and ANPEP; VEGF and c-MET; HER-2 and ERB3; HER-2 and BSG2;HER-2 and CDCP1; HER-2 and ANPEP; EGFR and CD64; EGFR and BSG2; EGFR andCDCP1; EGFR and ANPEP; IGF1R and PDGFR; IGF1R and VEGF; IGF1R and CD20;CD20 and CD74; CD20 and CD30; CD20 and DR4; CD20 and VEGFR2; CD20 andCD52; CD20 and CD4; HGF and c-MET; HGF and NRP1; HGF andphosphatidylserine; ErbB3 and IGF1R; ErbB3 and IGF1,2; c-Met and Her-2;c-Met and NRP1; c-Met and IGF1R; IGF1,2 and PDGFR; IGF1,2 and CD20;IGF1,2 and IGF1R; IGF2 and EGFR; IGF2 and HER2; IGF2 and CD20; IGF2 andVEGF; IGF2 and IGF1R; IGF1 and IGF2; PDGFRa and VEGFR2; PDGFRa and PLGF;PDGFRa and VEGF; PDGFRa and c-Met; PDGFRa and EGFR; PDGFRb and VEGFR2;PDGFRb and c-Met; PDGFRb and EGFR; RON and c-Met; RON and MTSP1; RON andMSP; RON and CDCP1; VGFR1 and PLGF; VGFR1 and RON; VGFR1 and EGFR;VEGFR2 and PLGF; VEGFR2 and NRP1; VEGFR2 and RON; VEGFR2 and DLL4;VEGFR2 and EGFR; VEGFR2 and ROBO4; VEGFR2 and CD55; LPA and S1 P; EPHB2and RON; CTLA4 and VEGF; CD3 and EPCAM; CD40 and IL6; CD40 and IGF; CD40and CD56; CD40 and CD70; CD40 and VEGFR1; CD40 and DR5; CD40 and DR4;CD40 and APRIL; CD40 and BCMA; CD40 and RANKL; CD28 and MAPG; CD80 andCD40; CD80 and CD30; CD80 and CD33; CD80 and CD74; CD80 and CD2; CD80and CD3; CD80 and CD19; CD80 and CD4; CD80 and CD52; CD80 and VEGF; CD80and DR5; CD80 and VEGFR2; CD22 and CD20; CD22 and CD80; CD22 and CD40;CD22 and CD23; CD22 and CD33; CD22 and CD74; CD22 and CD19; CD22 andDR5; CD22 and DR4; CD22 and VEGF; CD22 and CD52; CD30 and CD20; CD30 andCD22; CD30 and CD23; CD30 and CD40; CD30 and VEGF; CD30 and CD74; CD30and CD19; CD30 and DR5; CD30 and DR4; CD30 and VEGFR2; CD30 and CD52;CD30 and CD4; CD138 and RANKL; CD33 and FTL3; CD33 and VEGF; CD33 andVEGFR2; CD33 and CD44; CD33 and DR4; CD33 and DR5; DR4 and CD137; DR4and IGF1,2; DR4 and IGF1R; DR4 and DR5; DR5 and CD40; DR5 and CD137; DR5and CD20; DR5 and EGFR; DR5 and IGF1,2; DR5 and IGFR, DR5 and HER-2, andEGFR and DLL4. Other target combinations include one or more members ofthe EGF/erb-2/erb-3 family.

Other targets (one or more) involved in oncological diseases that themultispecific antibody analogs disclosed herein may bind include, butare not limited to those selected from the group consisting of: CD52,CD20, CD19, CD3, CD4, CD8, BMP6, IL12A, IL1A, IL1B, IL2, IL24, INHA,TNF, TNFSF10, BMP6, EGF, FGF1, FGF10, FGF11, FGF12, FGF13, FGF14, FGF16,FGF17, FGF18, FGF19, FGF2, FGF20, FGF21, FGF22, FGF23, FGF3, FGF4, FGF5,FGF6, FGF7, FGF8, FGF9, GRP, IGF1, IGF2, IL12A, IL1A, IL1B, IL2, INHA,TGFA, TGFB1, TGFB2, TGFB3, VEGF, CDK2, FGF10, FGF18, FGF2, FGF4, FGF7,IGF1R, IL2, BCL2, CD164, CDKN1A, CDKN1B, CDKN1C, CDKN2A, CDKN2B, CDKN2C,CDKN3, GNRH1, IGFBP6, IL1A, IL1B, ODZ1, PAWR, PLG, TGFBIII, AR, BRCA1,CDK3, CDK4, CDK5, CDK6, CDK7, CDK9, E2F1, EGFR, ENO1, ERBB2, ESR1, ESR2,IGFBP3, IGFBP6, IL2, INSL4, MYC, NOX5, NR6A1, PAP, PCNA, PRKCQ, PRKD1,PRL, TP53, FGF22, FGF23, FGF9, IGFBP3, IL2, INHA, KLK6, TP53, CHGB,GNRH1, IGF1, IGF2, INHA, INSL3, INSL4, PRL, KLK6, SHBG, NR1D1, NR1H3,NR113, NR2F6, NR4A3, ESR1, ESR2, NR0B1, NR0B2, NR1D2, NR1H2, NR1H4,NR112, NR2C1, NR2C2, NR2E1, NR2E3, NR2F1, NR2F2, NR3C1, NR3C2, NR4A1,NR4A2, NR5A1, NR5A2, NR6 μl, PGR, RARB, FGF1, FGF2, FGF6, KLK3, KRT1,APOC1, BRCA1, CHGA, CHGB, CLU, COL1A1, COL6A1, EGF, ERBB2, ERK8, FGF1,FGF10, FGF11, FGF13, FGF14, FGF16, FGF17, FGF18, FGF2, FGF20, FGF21,FGF22, FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, GNRH1, IGF1,IGF2, IGFBP3, IGFBP6, IL12A, IL1A, IL1B, IL2, IL24, INHA, INSL3, INSL4,KLK10, KLK12, KLK13, KLK14, KLK15, KLK3, KLK4, KLK5, KLK6, KLK9, MMP2,MMP9, MSMB, NTN4, ODZ1, PAP, PLAU, PRL, PSAP, SERPINA3, SHBG, TGFA,TIMP3, CD44, CDH1, CDH10, CDH19, CDH20, CDH7, CDH9, CDH1, CDH10, CDH13,CDH18, CDH19, CDH20, CDH7, CDH8, CDH9, ROBO2, CD44, ILK, ITGA1, APC,CD164, COL6A1, MTSS1, PAP, TGFBIII, AGR2, AIG1, AKAP1, AKAP2, CANT1,CAVI, CDH12, CLDN3, CLN3, CYB5, CYC1, DAB21P, DES, DNCL1, ELAC2, ENO2,ENO3, FASN, FLJ12584, FLJ25530, GAGEB1, GAGEC1, GGT1, GSTP1, HIP1,HUMCYT2A, IL29, K6HF, KAI1, KRT2A, MIB1, PART1, PATE, PCA3, PIAS2,PIK3CG, PPID, PR1, PSCA, SLC2A2, SLC33 μl, SLC43 μl, STEAP, STEAP2,TPM1, TPM2, TRPC6, ANGPT1, ANGPT2, ANPEP, ECGF1, EREG, FGF1, FGF2, FIGF,FLT1, JAG1, KDR, LAMAS, NRP1, NRP2, PGF, PLXDCI, STAB 1, VEGF, VEGFC,ANGPTL3, BA11, COL4A3, IL8, LAMAS, NRP1, NRP2, STAB 1, ANGPTL4, PECAM1,PF4, PROK2, SERPINF1, TNFAIP2, CCL11, CCL2, CXCL1, CXCL10, CXCL3, CXCL5,CXCL6, CXCL9, IFNA1, IFNB1, IFNG, IL1B, IL6, MDK, EDG1, EFNA1, EFNA3,EFNB2, EGF, EPHB4, FGFR3, HGF, IGF1, ITGB3, PDGFA, TEK, TGFA, TGFB1,TGFB2, TGFBR1, CCL2, CDH5, COL1A1, EDG1, ENG, ITGAV, ITGB3, THBS1,THBS2, BAD, BAG1, BCL2, CCNA1, CCNA2, CCND1, CCNE1, CCNE2, CDH1(E-cadherin), CDKN1B (p27Kip1), CDKN2A (p161NK4a), COL6A1, CTNNB1(b-catenin), CTSB (cathepsin B), ERBB2 (Her-2), ESR1, ESR2, F3 (TF),FOSL1 (FRA-1), GATA3, GSN (Gelsolin), IGFBP2, IL2RA, IL6, IL6R, IL6ST(glycoprotein 130), ITGA6 (a6 integrin), JUN, KLK5, KRT19, MAP2K7(c-Jun), MKI67 (Ki-67), NGFB (GF), NGFR, NME1 (M23A), PGR, PLAU (uPA),PTEN, SERPINB5 (maspin), SERPINE1 (PAI-1), TGFA, THBS1(thrombospondin-1), TIE (Tie-1), TNFRSF6 (Fas), TNFSF6 (FasL), TOP2A(topoisomerase Iia), TP53, AZGP1 (zinc-a-glycoprotein), BPAG1 (plectin),CDKN1A (p21Wap1/Cip1), CLDN7 (claudin-7), CLU (clusterin), ERBB2(Her-2), FGF1, FLRT1 (fibronectin), GABRP (GABAa), GNAS1, 1D2, ITGA6 (a6integrin), ITGB4 (b 4 integrin), KLF5 (GC Box BP), KRT19 (Keratin 19),KRTHB6 (hair-specific type II keratin), MACMARCKS, MT3(metallothionectin-111), MUC1 (mucin), PTGS2 (COX-2), RAC2 (p21Rac2),S100A2, SCGB1D2 (lipophilin B), SCGB2A1 (mammaglobin 2), SCGB2A2(mammaglobin 1), SPRR1B (Spr1), THBS1, THBS2, THBS4, and TNFAIP2 (B94),RON, c-Met, CD64, DLL4, PLGF, CTLA4, phophatidylserine, ROBO4, CD80,CD22, CD40, CD23, CD28, CD80, CD55, CD38, CD70, CD74, CD30, CD138, CD56,CD33, CD2, CD137, DR4, DRS, RANKL, VEGFR2, PDGFR, VEGFR1, MTSP1, MSP,EPHB2, EPHA1, EPHA2, EpCAM, PGE2, NKG2D, LPA, SIP, APRIL, BCMA, MAPG,FLT3, PDGFR alpha, PDGFR beta, ROR1, PSMA, PSCA, SCD1, and CD59.

Monoclonal antibody therapy has become an important therapeutic modalityfor treating autoimmune and inflammatory disorders (Chan & Carter, 2010,Nature Reviews Immunology 10:301-316; Reichert et al., 2005, NatureBiotechnology 23[9]:1073-1078). Many proteins have been implicated ingeneral autoimmune and inflammatory responses, and thus may be targetedby the inventive multispecific antibody analogs of the invention.Autoimmune and inflammatory targets include but are not limited to C5,CCL1 (1-309), CCL11 (eotaxin), CCL13 (mcp-4), CCL15 (MIP-1d), CCL16(HCC-4), CCL17 (TARC), CCL18 (PARC), CCL19, CCL2 (mcp-1), CCL20(MIP-3a), CCL21 (MIP-2), CCL23 (MPIF-1), CCL24 (MPIF-2/eotaxin-2), CCL25(TECK), CCL26, CCL3 (MIP-1a), CCL4 (MIP-1b), CCL5 (RANTES), CCL7(mcp-3), CCL8 (mcp-2), CXCL1, CXCL10 (IP-10), CXCL11 (1-TAC/IP-9),CXCL12 (SDF1), CXCL13, CXCL14, CXCL2, CXCL3, CXCL5 (ENA-78/LIX), CXCL6(GCP-2), CXCL9, IL13, IL8, CCL13 (mcp-4), CCR1, CCR2, CCR3, CCR4, CCR5,CCR6, CCR7, CCR8, CCR9, CX3CR1, IL8RA, XCR1 (CCXCR1), IFNA2, IL10, IL13,IL17C, IL1A, IL1B, IL1F10, IL1F5, IL1F6, IL1F7, IL1F8, IL1F9, IL22, IL5,IL8, IL9, LTA, LTB, MIF, SCYE1 (endothelial Monocyte-activatingcytokine), SPP1, TNF, TNFSF5, IFNA2, IL10RA, IL10RB, IL13, IL13RA1,IL5RA, IL9, IL9R, ABCF1, BCL6, C3, C4A, CEBPB, CRP, ICEBERG, IL1R1,IL1RN, IL8RB, LTB4R, TOLLIP, FADD, IRAK1, IRAK2, MYD88, NCK2, TNFAIP3,TRADD, TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, ACVR1, ACVR1B, ACVR2,ACVR2B, ACVRL1, CD28, CD3E, CD3G, CD3Z, CD69, CD80, CD86, CNR1, CTLA4,CYSLTR1, FCER1A, FCER2, FCGR3A, GPR44, HAVCR2, OPRD1, P2RX7, TLR2, TLR3,TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, BLR1, CCL1, CCL2, CCL3, CCL4,CCL5, CCL7, CCL8, CCL11, CCL13, CCL15, CCL16, CCL17, CCL18, CCL19,CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCR1, CCR2, CCR3, CCR4, CCR5,CCR6, CCR7, CCR8, CCR9, CX3CL1, CX3CR1, CXCL1, CXCL2, CXCL3, CXCL5,CXCL6, CXCL10, CXCL11, CXCL12, CXCL13, CXCR4, GPR2, SCYE1, SDF2, XCL1,XCL2, XCR1, AMH, AMHR2, BMPR1A, BMPR1B, BMPR2, C19orf10 (IL27w), CERT,CSF1, CSF2, CSF3, DKFZp451J0118, FGF2, GFI1, IFNA1, IFNB1, IFNG, IGF1,IL1A, IL1B, IL1R1, IL1R2, IL2, IL2RA, IL2RB, IL2RG, IL3, IL4, IL4R, IL5,IL5RA, IL6, IL6R, IL6ST, IL7, IL8, IL8RA, IL8RB, IL9, IL9R, IL10,IL10RA, IL10RB, IL11, IL12RA, IL12A, IL12B, IL12RB1, IL12RB2, IL13,IL13RA1, IL13RA2, IL15, IL15RA, IL16, IL17, IL17R, IL18, IL18R1, IL19,IL20, KITLG, LEP, LTA, LTB, LTB4R, LTB4R2, LTBR, MIF, NPPB, PDGFB,TBX21, TDGF1, TGFA, TGFB1, TGFB1I1, TGFB2, TGFB3, TGFB1, TGFBR1, TGFBR2,TGFBR3, TH1L, TNF, TNFRSF1A, TNFRSF1B, TNFRSF7, TNFRSF8, TNFRSF9,TNFRSF11A, TNFRSF21, TNFSF4, TNFSF5, TNFSF6, TNFSF11, VEGF, ZFPM2, andRNF110 (ZNF144).

Exemplary co-targets for autoimmune and inflammatory disorders includebut are not limited to IL-1 and TNFalpha, IL-6 and TNFalpha, IL-6 andIL-1, IgE and IL-13, IL-1 and IL-13, IL-4 and IL-13, IL-5 and IL-13,IL-9 and IL-13, CD19 and FcγRIIb, and CD79 and FcγRIIb.

Multispecific antibody analogs of the invention with specificity for thefollowing pairs of targets to treat inflammatory disease arecontemplated: TNF and IL-17A; TNF and RANKL; TNF and VEGF; TNF and SOST;TNF and DKK; TNF and alphaVbeta3; TNF and NGF; TNF and IL-23p19; TNF andIL-6; TNF and SOST; TNF and IL-6R; TNF and CD-20; IgE and IL-13; IL-13and IL23p19; IgE and IL-4; IgE and IL-9; IgE and IL-9; IgE and IL-13;IL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-9; IL-13 and IL-9; IL-13and IL-4; IL-13 and IL-23p19; IL-13 and IL-9; IL-6R and VEGF; IL-6R andIL-17A; IL-6R and RANKL; IL-17A and IL-1 beta; IL-1 beta and RANKL;IL-1beta and VEGF; RANKL and CD-20; IL-1alpha and IL-1 beta; IL-1 alphaand IL-1beta.

Pairs of targets that the multispecific antibody analogs describedherein can bind and be useful to treat asthma may be determined. In anembodiment, such targets include, but are not limited to, IL-13 and IL-1beta, since IL-1 beta is also implicated in inflammatory response inasthma; IL-13 and cytokines and chemokines that are involved ininflammation, such as IL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-5;IL-13 and IL-25; IL-13 and TARC; IL-13 and MDC; IL-13 and MIF; IL-13 andTGF-β; IL-13 and LHR agonist; IL-13 and CL25; IL-13 and SPRR2a; IL-13and SPRR2b; and IL-13 and ADAMS. The inventive multispecific antibodyanalogs herein may have specificity for one or more targets involved inasthma selected from the group consisting of CSF1 (MCSF), CSF2 (GM-CSF),CSF3 (GCSF), FGF2, IFNA1, IFNB1, IFNG, histamine and histaminereceptors, IL1A, IL1B, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10,IL11, IL12A, IL12B, IL13, IL14, IL15, IL16, IL17, IL18, IL19, KITLG,PDGFB, IL2RA, IL4R, IL5RA, IL8RA, IL8RB, IL12RB1, IL12RB2, IL13RA1,IL13RA2, IL18R1, TSLP, CCLi, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL13,CCL17, CCL18, CCL19, CCL20, CCL22, CCL24, CX3CL1, CXCL1, CXCL2, CXCL3,XCLi, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CX3CR1, GPR2, XCR1, FOS,GATA3, JAK1, JAK3, STATE, TBX21, TGFB1, TNF, TNFSF6, YY1, CYSLTR1,FCER1A, FCER2, LTB4R, TB4R2, LTBR, and Chitinase.

Pairs of targets involved in rheumatoid arthritis (RA) may beco-targeted by the invention, including but not limited to TNF andIL-18; TNF and IL-12; TNF and IL-23; TNF and 1L-1beta; TNF and MIF; TNFand IL-17; and TNF and IL-15.

Antigens that may be targeted in order to treat systemic lupuserythematosus (SLE) by the inventive multispecific antibody analogsherein include but are not limited to CD-20, CD-22, CD-19, CD28, CD4,CD80, HLA-DRA, IL10, IL2, IL4, TNFRSF5, TNFRSF6, TNFSF5, TNFSF6, BLR1,HDAC4, HDAC5, HDAC7A, HDAC9, ICOSL, IGBP1, MS4A1, RGSI, SLA2, CD81,IFNB1, IL10, TNFRSF5, TNFRSF7, TNFSF5, AICDA, BLNK, GALNAC4S-6ST, HDAC4,HDAC5, HDAC7A, HDAC9, IL10, IL11, IL4, INHA, INHBA, KLF6, TNFRSF7, CD28,CD38, CD69, CD80, CD83, CD86, DPP4, FCER2, IL2RA, TNFRSF8, TNFSF7, CD24,CD37, CD40, CD72, CD74, CD79A, CD79B, CR2, ILIR2, ITGA2, ITGA3, MS4A1,ST6GALI, CDIC, CHSTIO, HLA-A, HLA-DRA, and NT5E; CTLA4, B7.1, B7.2,BIyS, BAFF, C5, IL-4, IL-6, IL-10, IFN-α, and TNF-α.

The inventive multispecific antibody analogs herein may target antigensfor the treatment of multiple sclerosis (MS), including but not limitedto IL-12, TWEAK, IL-23, CXCL13, CD40, CD40L, IL-18, VEGF, VLA-4, TNF,CD45RB, CD200, IFNgamma, GM-CSF, FGF, C5, CD52, and CCR2. An embodimentincludes co-engagement of anti-IL-12 and TWEAK for the treatment of MS.

One aspect of the invention pertains to inventive multispecific antibodyanalogs capable of binding one or more targets involved in sepsis, in anembodiment two targets, selected from the group consisting TNF, IL-1,MIF, IL-6, IL-8, IL-18, IL-12, IL-23, FasL, LPS, Toll-like receptors,TLR-4, tissue factor, MIP-2, ADORA2A, CASP1, CASP4, IL-10, IL-1B, NFκB1,PROC, TNFRSFIA, CSF3, CCR3, ILIRN, MIF, NFκB1, PTAFR, TLR2, TLR4, GPR44,HMOX1, midkine, IRAK1, NFκB2, SERPINA1, SERPINE1, and TREM1.

In some cases, inventive multispecific antibody analogs herein may bedirected against antigens for the treatment of infectious diseases.

The inventive multispecific antibody analogs may be screened using avariety of in vitro methods, including but not limited to those that usebinding assays, cell-based assays, and selection technologies.Automation and high-throughput screening technologies may be utilized inthe screening procedures. Screening may employ the use of a fusionpartner or label. The use of fusion partners has been discussed above.By “labeled” herein is meant that the inventive multispecific antibodyanalogs disclosed herein have one or more elements, isotopes, orchemical compounds attached to enable the detection in a screen. Ingeneral, labels fall into three classes: a) immune labels, which may bean epitope incorporated as a fusion partner that is recognized by anantibody, b) isotopic labels, which may be radioactive or heavyisotopes, and c) small molecule labels, which may include fluorescentand colorimetric dyes, or molecules such as biotin that enable otherlabeling methods. Labels may be incorporated into the compound at anyposition and may be incorporated in vitro or in vivo during proteinexpression.

In certain embodiments, the functional and/or biophysical properties ofthe inventive multispecific antibody analogs are screened in an in vitroassay. In vitro assays may allow a broad dynamic range for screeningproperties of interest. Particularly relevant for the present invention,the inventive multispecific antibody analogs may be tested for theiraffinity for one or more antigens. Properties that may be screenedinclude but are not limited to stability, solubility, and affinity forFc ligands, for example FcγRs. Multiple properties may be screenedsimultaneously or individually. Proteins may be purified or unpurified,depending on the requirements of the assay. In one embodiment, thescreen is a qualitative or quantitative binding assay for binding ofinventive multispecific antibody analogs to a protein or nonproteinmolecule that is known or thought to bind the inventive multispecificantibody analog. In one embodiment, the screen is a binding assay formeasuring binding to the target antigen. In an alternate embodiment, thescreen is an assay for binding of inventive multispecific antibodyanalogs to an Fc ligand, including but are not limited to the family ofFcγRs, the neonatal receptor FcRn, the complement protein C1q, and thebacterial proteins A and G. Said Fc ligands may be from any organism. Inone embodiment, Fc ligands are from humans, mice, rats, rabbits, and/ormonkeys. Binding assays can be carried out using a variety of methodsknown in the art, including but not limited to FRET (FluorescenceResonance Energy Transfer) and BRET (Bioluminescence Resonance EnergyTransfer)-based assays, AlphaScreen™ (Amplified Luminescent ProximityHomogeneous Assay), Scintillation Proximity Assay, ELISA (Enzyme-LinkedImmunosorbent Assay), SPR (Surface Plasmon Resonance, also known asBIACORE®), isothermal titration calorimetry, differential scanningcalorimetry, gel electrophoresis, and chromatography including gelfiltration. These and other methods may take advantage of some fusionpartner or label of the inventive multispecific antibody analog. Assaysmay employ a variety of detection methods including but not limited tochromogenic, fluorescent, luminescent, or isotopic labels.

The biophysical properties of the inventive multispecific antibodyanalogs, for example stability and solubility, may be tested using avariety of methods known in the art. Protein stability may be determinedby measuring the thermodynamic equilibrium between folded and unfoldedstates. For example, inventive multispecific antibody analogs disclosedherein may be unfolded using chemical denaturant, heat, or pH, and thistransition may be monitored using methods including but not limited tocircular dichroism spectroscopy, fluorescence spectroscopy, absorbancespectroscopy, NMR spectroscopy, calorimetry, and proteolysis. As will beappreciated by those skilled in the art, the kinetic parameters of thefolding and unfolding transitions may also be monitored using these andother techniques. The solubility and overall structural integrity of aninventive multispecific antibody analog may be quantitatively orqualitatively determined using a wide range of methods that are known inthe art. Methods which may find use for characterizing the biophysicalproperties of inventive multispecific antibody analogs disclosed hereininclude gel electrophoresis, isoelectric focusing, capillaryelectrophoresis, chromatography such as size exclusion chromatography,ion-exchange chromatography, and reversed-phase high performance liquidchromatography, peptide mapping, oligosaccharide mapping, massspectrometry, ultraviolet absorbance spectroscopy, fluorescencespectroscopy, circular dichroism spectroscopy, isothermal titrationcalorimetry, differential scanning calorimetry, analyticalultra-centrifugation, dynamic light scattering, proteolysis, andcross-linking, turbidity measurement, filter retardation assays,immunological assays, fluorescent dye binding assays, protein-stainingassays, microscopy, and detection of aggregates via ELISA or otherbinding assay. Structural analysis employing X-ray crystallographictechniques and NMR spectroscopy may also find use. In one embodiment,stability and/or solubility may be measured by determining the amount ofprotein solution after some defined period of time. In this assay, theprotein may or may not be exposed to some extreme condition, for exampleelevated temperature, low pH, or the presence of denaturant. Becausefunction typically requires a stable, soluble, and/orwell-folded/structured protein, the aforementioned functional andbinding assays also provide ways to perform such a measurement. Forexample, a solution comprising an inventive multispecific antibodyanalog could be assayed for its ability to bind target antigen, thenexposed to elevated temperature for one or more defined periods of time,then assayed for antigen binding again. Because unfolded and aggregatedprotein is not expected to be capable of binding antigen, the amount ofactivity remaining provides a measure of the inventive multispecificantibody analog's stability and solubility.

In certain embodiments, the inventive multispecific antibody analogs maybe tested using one or more cell-based or in vitro assays. For suchassays, inventive multispecific antibody analogs, purified orunpurified, are typically added exogenously such that cells are exposedto inventive multispecific antibody analogs described herein. Theseassays are typically, but not always, based on the biology of theability of the inventive multispecific antibody analog to bind to thetarget antigen and mediate some biochemical event, for example effectorfunctions like cellular lysis, phagocytosis, ligand/receptor bindinginhibition, inhibition of growth and/or proliferation, inhibition ofcalcium release and/or signaling, apoptosis and the like. Such assaysoften involve monitoring the response of cells to inventivemultispecific antibody analog, for example cell survival, cell death,cellular phagocytosis, cell lysis, change in cellular morphology, ortranscriptional activation such as cellular expression of a natural geneor reporter gene. For example, such assays may measure the ability ofinventive multispecific antibody analogs to elicit cell killing, forexample ADCC, ADCP, and CDC. Assays that measure cellular killing thatis mediated by co-engagement of antigens are particularly relevant forthe invention. For some assays additional cells or components, that isin addition to the target cells, may need to be added, for example serumcomplement, or effector cells such as peripheral blood monocytes(PBMCs), NK cells, macrophages, T cells, and the like. Such additionalcells may be from any organism, e.g., humans, mice, rat, rabbit, andmonkey. Crosslinked or monomeric antibodies may cause apoptosis ofcertain cell lines expressing the antibody's target antigen, or they maymediate attack on target cells by immune cells which have been added tothe assay. Methods for monitoring cell death or viability are known inthe art, and include the use of dyes, fluorophores, immunochemical,cytochemical, and radioactive reagents. For example, caspase assays orannexin-flourconjugates may enable apoptosis to be measured, and uptakeor release of radioactive substrates (e.g. Chromium-51 release assays)or the metabolic reduction of fluorescent dyes such as alamar blue mayenable cell growth, proliferation or activation to be monitored. In oneembodiment, the DELFIA EuTDA-based cytotoxicity assay (Perkin Elmer,Mass.) is used. Alternatively, dead or damaged target cells may bemonitored by measuring the release of one or more natural intracellularproteins, for example lactate dehydrogenase. Transcriptional activationmay also serve as a method for assaying function in cell-based assays.In this case, response may be monitored by assaying for natural genes orproteins which may be upregulated or down-regulated, for example therelease of certain interleukins may be measured, or alternativelyreadout may be via a luciferase or GFP-reporter construct. Cell-basedassays may also involve the measure of morphological changes of cells asa response to the presence of an inventive multispecific antibodyanalog. Cell types for such assays may be prokaryotic or eukaryotic, anda variety of cell lines that are known in the art may be employed.Alternatively, cell-based screens are performed using cells that havebeen transformed or transfected with nucleic acids encoding theinventive multispecific antibody analogs.

The biological properties of the inventive multispecific antibodyanalogs disclosed herein may be characterized in cell, tissue, and wholeorganism experiments. As is known in the art, drugs are often tested inanimals, including but not limited to mice, rats, rabbits, dogs, cats,pigs, and monkeys, in order to measure a drug's efficacy for treatmentagainst a disease or disease model, or to measure a drug'spharmacokinetics, toxicity, and other properties. Said animals may bereferred to as disease models. With respect to the inventivemultispecific antibody analogs disclosed herein, a particular challengearises when using animal models to evaluate the potential for in-humanefficacy of candidate polypeptides—this is due, at least in part, to thefact that inventive multispecific antibody analogs that have a specificeffect on the affinity for a human Fc receptor may not have a similaraffinity effect with the orthologous animal receptor. These problems canbe further exacerbated by the inevitable ambiguities associated withcorrect assignment of true orthologues (Mechetina et al., 2002,Immunogenetics 54:463-468), and the fact that some orthologues simply donot exist in the animal. Therapeutics are often tested in mice,including but not limited to nude mice, Rag-deficient mice, SCID mice,xenograft mice, and transgenic mice (including knockins and knockouts).For example, an inventive multispecific antibody analog of the presentinvention that is intended as an anti-cancer therapeutic may be testedin a mouse cancer model, for example a xenograft mouse. In this method,a tumor or tumor cell line is grafted onto or injected into a mouse, andsubsequently the mouse is treated with the therapeutic to determine theability of the drug to reduce or inhibit cancer growth and metastasis.Therapeutic inventive multispecific antibody analogs herein can betested in mouse strains NZB, NOD, BXSB, MRL/Ipr, K/BxN and transgenics(including knockins and knockouts). Such mice can develop variousautoimmune conditions that resemble human organ specific, systemicautoimmune or inflammatory disease pathologies such as systemic lupuserythematosus (SLE) and rheumatoid arthritis (RA). For example, aninventive multispecific antibody analog disclosed herein intended forautoimmune diseases may be tested in such mouse models by treating themice to determine the ability of the inventive multispecific antibodyanalog to reduce or inhibit the development of the disease pathology.Because of the incompatibility between the mouse and human Fey receptorsystem, an alternative approach is to use a murine SCID model in whichimmune deficient mice are engrafted with human PBLs or PBMCs(huPBL-SCID, huPBMC-SCID) providing a semi-functional human immunesystem with human effector cells and Fc receptors. Other organisms,e.g., mammals, may also be used for testing. For example, because oftheir genetic similarity to humans, monkeys can be suitable therapeuticmodels, and thus may be used to test the efficacy, toxicity,pharmacokinetics, or other property of the inventive multispecificantibody analogs disclosed herein. Tests of the inventive multispecificantibody analogs disclosed herein in humans are ultimately required forapproval as drugs, and thus of course these experiments arecontemplated. Thus the inventive multispecific antibody analogsdisclosed herein may be tested in humans to determine their therapeuticefficacy, toxicity, pharmacokinetics, and/or other clinical properties.

In some embodiments, inventive multispecific antibody analogs disclosedherein may be assessed for efficacy in clinically relevant animal modelsof various human diseases. In many cases, relevant models includevarious transgenic animals for specific antigens and receptors.

In certain embodiments, the testing of inventive multispecific antibodyanalogs may include study of efficacy in primates (e.g. cynomolgusmonkey model) to facilitate the evaluation of depletion of specifictarget cells harboring the target antigen. Additional primate modelsinclude but are not limited to use of the rhesus monkey to assessinventive multispecific antibody analogs in therapeutic studies ofautoimmune, transplantation and cancer.

Toxicity studies are performed to determine drug related-effects thatcannot be evaluated in standard pharmacology profiles, or occur onlyafter repeated administration of the agent. Most toxicity tests areperformed in two species—a rodent and a non-rodent—to ensure that anyunexpected adverse effects are not overlooked before new therapeuticentities are introduced into man. In general, these models may measure avariety of toxicities including genotoxicity, chronic toxicity,immunogenicity, reproductive/developmental toxicity and carcinogenicity.Included within the aforementioned parameters are standard measurementof food consumption, bodyweight, antibody formation, clinical chemistry,and macro- and microscopic examination of standard organs/tissues (e.g.cardiotoxicity). Additional parameters of measurement are injection sitetrauma and the measurement of neutralizing antibodies, if any.Traditionally, monoclonal antibody therapeutics, naked or conjugated, isevaluated for cross-reactivity with normal tissues,immunogenicity/antibody production, conjugate or linker toxicity and“bystander” toxicity of radiolabelled species. Nonetheless, such studiesmay have to be individualized to address specific concerns and followingthe guidance set by ICH S6 (Safety studies for biotechnologicalproducts, also noted above). As such, the general principles are thatthe products are sufficiently well characterized,impurities/contaminants have been removed, that the test material iscomparable throughout development, and that GLP compliance ismaintained.

The pharmacokinetics (PK) of the inventive multispecific antibodyanalogs disclosed herein may be studied in a variety of animal systems,with the most relevant being non-human primates such as the cynomolgusand rhesus monkeys. Single or repeated i.v./s.c. administrations over adose range of 6000-fold (0.05-300 mg/kg) can be evaluated for half-life(days to weeks) using plasma concentration and clearance. Volume ofdistribution at a steady state and level of systemic absorbance can alsobe measured. Examples of such parameters of measurement generallyinclude maximum observed plasma concentration (Cmax), the time to reachCmax (Tmax), the area under the plasma concentration-time curve fromtime 0 to infinity [AUC(0-inf] and apparent elimination half-life(T1/2). Additional measured parameters could include compartmentalanalysis of concentration-time data obtained following i.v.administration and bioavailability.

Pharmacodynamic studies may include, but are not limited to, targetingspecific cells or blocking signaling mechanisms, measuring inhibition ofantigen-specific antibodies etc. The inventive multispecific antibodyanalogs disclosed herein may target particular effector cell populationsand thereby be direct drugs to induce certain activities to improvepotency or to increase penetration into a particularly favorablephysiological compartment. Such pharmacodynamic effects may bedemonstrated in animal models or in humans.

The inventive multispecific antibody analogs disclosed herein may finduse in a wide range of products. In one embodiment an inventivemultispecific antibody analog disclosed herein comprise a therapeutic, adiagnostic, or a research reagent. The inventive multispecific antibodyanalogs may find use in a composition that is monoclonal or polyclonal.The inventive multispecific antibody analogs disclosed herein may beused for therapeutic purposes. As will be appreciated by those in theart, the inventive multispecific antibody analogs disclosed herein maybe used for any therapeutic purpose that antibodies, Fc fusions, and thelike may be used for. The inventive multispecific antibody analogs maybe administered to a patient to treat disorders including but notlimited to cancer, infectious diseases, autoimmune and inflammatorydiseases.

A “patient” for the purposes disclosed herein includes both humans andother animals, e.g., other mammals. Thus the inventive multispecificantibody analogs disclosed herein have both human therapy and veterinaryapplications. The term “treatment” or “treating” as disclosed herein ismeant to include therapeutic treatment, as well as prophylactic, orsuppressive measures for a disease or disorder. Thus, for example,successful administration of an inventive multispecific antibody analogprior to onset of the disease results in treatment of the disease. Asanother example, successful administration of an optimized inventivemultispecific antibody analog after clinical manifestation of thedisease to combat the symptoms of the disease comprises treatment of thedisease. “Treatment” and “treating” also encompasses administration ofan optimized inventive multispecific antibody analog after theappearance of the disease in order to eradicate the disease. Successfuladministration of an agent after onset and after clinical symptoms havedeveloped, with possible abatement of clinical symptoms and perhapsamelioration of the disease, comprises treatment of the disease. Those“in need of treatment” include mammals already having the disease ordisorder, as well as those prone to having the disease or disorder,including those in which the disease or disorder is to be prevented.

In one embodiment, the inventive multispecific antibody analogsdisclosed herein are administered to a patient having a diseaseinvolving inappropriate expression of a protein or other molecule.Within the scope disclosed herein this is meant to include diseases anddisorders characterized by aberrant proteins, due for example toalterations in the amount of a protein present, protein localization,posttranslational modification, conformational state, the presence of amutant or pathogen protein, etc. Similarly, the disease or disorder maybe characterized by alterations molecules including but not limited topolysaccharides and gangliosides. An overabundance may be due to anycause, including but not limited to overexpression at the molecularlevel, prolonged or accumulated appearance at the site of action, orincreased activity of a protein relative to normal. Included within thisdefinition are diseases and disorders characterized by a reduction of aprotein. This reduction may be due to any cause, including but notlimited to reduced expression at the molecular level, shortened orreduced appearance at the site of action, mutant forms of a protein, ordecreased activity of a protein relative to normal. Such anoverabundance or reduction of a protein can be measured relative tonormal expression, appearance, or activity of a protein, and saidmeasurement may play an important role in the development and/orclinical testing of the inventive multispecific antibody analogsdisclosed herein.

The inventive multispecific antibody analogs herein may be used to treatcancer. By “cancer” and “cancerous” herein refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto carcinoma, lymphoma, blastoma, sarcoma (including liposarcoma),neuroendocrine tumors, mesothelioma, schwanoma, meningioma,adenocarcinoma, melanoma, and leukemia or lymphoid malignancies.

More particular examples of such cancers include hematologicmalignancies, such as Hodgkin's lymphoma; non-Hodgkin's lymphomas(Burkitt's lymphoma, small lymphocytic lymphoma/chronic lymphocyticleukemia, mycosis fungoides, mantle cell lymphoma, follicular lymphoma,diffuse large B-cell lymphoma, marginal zone lymphoma, hairy cellleukemia and lymphoplasmacytic leukemia), tumors of lymphocyte precursorcells, including B-cell acute lymphoblastic leukemia/lymphoma, andT-cell acute lymphoblastic leukemia/lymphoma, thymoma, tumors of themature T and NK cells, including peripheral T-cell leukemias, adultT-cell leukemia/T-cell lymphomas and large granular lymphocyticleukemia, Langerhans cell histocytosis, myeloid neoplasias such as acutemyelogenous leukemias, including AML with maturation, AML withoutdifferentiation, acute promyelocytic leukemia, acute myelomonocyticleukemia, and acute monocytic leukemias, myelodysplastic syndromes, andchronic myeloproliferative disorders, including chronic myelogenousleukemia; tumors of the central nervous system such as glioma,glioblastoma, neuroblastoma, astrocytoma, medulloblastoma, ependymoma,and retinoblastoma; solid tumors of the head and neck (e.g.nasopharyngeal cancer, salivary gland carcinoma, and esophagael cancer),lung (e.g. small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung and squamous carcinoma of the lung),digestive system (e.g. gastric or stomach cancer includinggastrointestinal cancer, cancer of the bile duct or biliary tract, coloncancer, rectal cancer, colorectal cancer, and anal carcinoma),reproductive system (e.g. testicular, penile, or prostate cancer,uterine, vaginal, vulval, cervical, ovarian, and endometrial cancer),skin (e.g. melanoma, basal cell carcinoma, squamous cell cancer, actinickeratosis), liver (e.g. liver cancer, hepatic carcinoma, hepatocellularcancer, and hepatoma), bone (e.g. osteoclastoma, and osteolytic bonecancers) additional tissues and organs (e.g. pancreatic cancer, bladdercancer, kidney or renal cancer, thyroid cancer, breast cancer, cancer ofthe peritoneum, and Kaposi's sarcoma), and tumors of the vascular system(e.g. angiosarcoma and hemagiopericytoma).

The inventive multispecific antibody analogs disclosed herein may beused to treat autoimmune diseases. By “autoimmune diseases” hereininclude allogenic islet graft rejection, alopecia areata, ankylosingspondylitis, antiphospholipid syndrome, autoimmune Addison's disease,antineutrophil cytoplasmic autoantibodies (ANCA), autoimmune diseases ofthe adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis,autoimmune myocarditis, autoimmune neutropenia, autoimmune oophoritisand orchitis, autoimmune thrombocytopenia, autoimmune urticaria,Behcet's disease, bullous pemphigoid, cardiomyopathy, Castleman'ssyndrome, celiac spruce-dermatitis, chronic fatigue immune dysfunctionsyndrome, chronic inflammatory demyelinating polyneuropathy,Churg-Strauss syndrome, cicatrical pemphigoid, CREST syndrome, coldagglutinin disease, Crohn's disease, dermatomyositis, discoid lupus,essential mixed cryoglobulinemia, factor VIII deficiency,fibromyalgia-fibromyositis, glomerulonephritis, Grave's disease,Guillain-Barre, Goodpasture's syndrome, graft-versus-host disease(GVHD), Hashimoto's thyroiditis, hemophilia A, idiopathic pulmonaryfibrosis, idiopathic thrombocytopenia purpura (ITP), IgA neuropathy, IgMpolyneuropathies, immune mediated thrombocytopenia, juvenile arthritis,Kawasaki's disease, lichen plantus, lupus erthematosis, Meniere'sdisease, mixed connective tissue disease, multiple sclerosis, type 1diabetes mellitus, myasthenia gravis, pemphigus vulgaris, perniciousanemia, polyarteritis nodosa, polychrondritis, polyglandular syndromes,polymyalgia rheumatica, polymyositis and dermatomyositis, primaryagammaglobinulinemia, primary biliary cirrhosis, psoriasis, psoriaticarthritis, Reynauld's phenomenon, Reiter's syndrome, rheumatoidarthritis, sarcoidosis, scleroderma, Sjorgen's syndrome, solid organtransplant rejection, stiff-man syndrome, systemic lupus erythematosus,takayasu arteritis, temporal arteristis/giant cell arteritis, thromboticthrombocytopenia purpura, ulcerative colitis, uveitis, vasculitides suchas dermatitis herpetiformis vasculitis, vitiligo, and Wegner'sgranulomatosis.

The inventive multispecific antibody analogs disclosed herein may beused to treat inflammatory disorders. By “inflammatory disorders” hereininclude acute respiratory distress syndrome (ARDS), acute septicarthritis, adjuvant arthritis, juvenile idiopathic arthritis, allergicencephalomyelitis, allergic rhinitis, allergic vasculitis, allergy,asthma, atherosclerosis, chronic inflammation due to chronic bacterialor viral infections, chronic obstructive pulmonary disease (COPD),coronary artery disease, encephalitis, inflammatory bowel disease,inflammatory osteolysis, inflammation associated with acute and delayedhypersensitivity reactions, inflammation associated with tumors,peripheral nerve injury or demyelinating diseases, inflammationassociated with tissue trauma such as burns and ischemia, inflammationdue to meningitis, multiple organ injury syndrome, pulmonary fibrosis,sepsis and septic shock, Stevens-Johnson syndrome, undifferentiatedarthropy, and undifferentiated spondyloarthropathy.

Some autoimmune and inflammatory diseases that may be targeted by theinventive multispecific antibody analogs disclosed herein includeSystemic Lupus Erythematosus, Rheumatoid arthritis, Sjogren's syndrome,Multiple sclerosis, Idiopathic thrombocytopenic purpura (ITP), Gravesdisease, Inflammatory bowel disease, Psoriasis, Type I diabetes, andAsthma.

The inventive multispecific antibody analogs herein may be used to treatinfectious diseases. By “infectious diseases” herein include diseasescaused by pathogens such as viruses, bacteria, fungi, protozoa, andparasites. Infectious diseases may be caused by viruses includingadenovirus, cytomegalovirus, dengue, Epstein-Barr, hanta, hepatitis A,hepatitis B, hepatitis C, herpes simplex type I, herpes simplex type II,human immunodeficiency virus, (HIV), human papilloma virus (HPV),influenza, measles, mumps, papova virus, polio, respiratory syncytialvirus, rinderpest, rhinovirus, rotavirus, rubella, SARS virus, smallpox,viral meningitis, and the like. Infectious diseases may also be causedby bacteria including Bacillus antracis, Borrelia burgdorferi,Campylobacter jejuni, Chlamydia trachomatis, Clostridium botulinum,Clostridium tetani, Diptheria, E. coli, Legionella, Helicobacter pylori,Mycobacterium rickettsia, Mycoplasma nesisseria, Pertussis, Pseudomonasaeruginosa, S. pneumonia, Streptococcus, Staphylococcus, Vibriacholerae, Yersinia pestis, and the like. Infectious diseases may also becaused by fungi such as Aspergillus fumigatus, Blastomyces dermatitidis,Candida albicans, Coccidioides immitis, Cryptococcus neoformans,Histoplasma capsulatum, Penicillium marneffei, and the like. Infectiousdiseases may also be caused by protozoa and parasites such as chlamydia,kokzidioa, leishmania, malaria, rickettsia, trypanosoma, and the like.

Furthermore, inventive multispecific antibody analogs disclosed hereinmay be used to prevent or treat additional conditions including but notlimited to heart conditions such as congestive heart failure (CHF),myocarditis and other conditions of the myocardium; skin conditions suchas rosecea, acne, and eczema; bone and tooth conditions such as boneloss, osteoporosis, Paget's disease, Langerhans' cell histiocytosis,periodontal disease, disuse osteopenia, osteomalacia, monostotic fibrousdysplasia, polyostotic fibrous dysplasia, bone metastasis, bone painmanagement, humoral malignant hypercalcemia, periodontal reconstruction,spinal cord injury, and bone fractures; metabolic conditions such asGaucher's disease; endocrine conditions such as Cushing's syndrome; andneurological and neurodegenerative conditions such as Alzheimer'sdisease.

Pharmaceutical compositions are contemplated wherein an inventivemultispecific antibody analog disclosed herein and one or moretherapeutically active agents are formulated. Formulations of theinventive multispecific antibody analogs disclosed herein are preparedfor storage by mixing said inventive multispecific antibody analoghaving the desired degree of purity with optional pharmaceuticallyacceptable carriers, excipients or stabilizers (Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed., 1980), in the formof lyophilized formulations or aqueous solutions. Acceptable carriers,excipients, or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrate,acetate, and other organic acids; antioxidants including ascorbic acidand methionine; preservatives (such as octadecyldimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzethoniumchloride; phenol, butyl orbenzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrins; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; sweeteners andother flavoring agents; fillers such as microcrystalline cellulose,lactose, corn and other starches; binding agents; additives; coloringagents; salt-forming counter-ions such as sodium; metal complexes (e.g.Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™,PLURONICS™ or polyethylene glycol (PEG). In one embodiment, thepharmaceutical composition that comprises the inventive multispecificantibody analog disclosed herein may be in a water-soluble form, such asbeing present as pharmaceutically acceptable salts, which is meant toinclude both acid and base addition salts. “Pharmaceutically acceptableacid addition salt” refers to those salts that retain the biologicaleffectiveness of the free bases and that are not biologically orotherwise undesirable, formed with inorganic acids such as hydrochloricacid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid andthe like, and organic acids such as acetic acid, propionic acid,glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid,succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid,cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid and the like. “Pharmaceuticallyacceptable base addition salts” include those derived from inorganicbases such as sodium, potassium, lithium, ammonium, calcium, magnesium,iron, zinc, copper, manganese, aluminum salts and the like. Someembodiments include at least one of the ammonium, potassium, sodium,calcium, and magnesium salts. Salts derived from pharmaceuticallyacceptable organic non-toxic bases include salts of primary, secondary,and tertiary amines, substituted amines including naturally occurringsubstituted amines, cyclic amines and basic ion exchange resins, such asisopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, and ethanolamine. The formulations to be used for invivo administration may be sterile. This is readily accomplished byfiltration through sterile filtration membranes or other methods.

The inventive multispecific antibody analogs disclosed herein may alsobe formulated as immunoliposomes. A liposome is a small vesiclecomprising various types of lipids, phospholipids and/or surfactant thatis useful for delivery of a therapeutic agent to a mammal. Liposomescontaining the inventive multispecific antibody analog are prepared bymethods known in the art. The components of the liposome are commonlyarranged in a bilayer formation, similar to the lipid arrangement ofbiological membranes. Particularly useful liposomes can be generated bythe reverse phase evaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter.

An inventive multispecific antibody analog and other therapeuticallyactive agents may also be entrapped in microcapsules prepared by methodsincluding but not limited to coacervation techniques, interfacialpolymerization (for example using hydroxymethylcellulose orgelatin-microcapsules, or poly-(methylmethacylate) microcapsules),colloidal drug delivery systems (for example, liposomes, albuminmicrospheres, microemulsions, nano-particles and nanocapsules), andmacroemulsions. Such techniques are disclosed in Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed., 1980.Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymer, which matrices are in the form of shaped articles,e.g. films, or microcapsules. Examples of sustained-release matricesinclude polyesters, hydrogels (for examplepoly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides,copolymers of L-glutamic acid and gamma ethyl-L-glutamate,non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolicacid copolymers such as the Lupron Depot® (which are injectablemicrospheres composed of lactic acid-glycolic acid copolymer andleuprolide acetate), poly-D-(−)-3-hydroxybutyric acid, and ProLease®(commercially available from Alkermes), which is a microsphere-baseddelivery system composed of the desired bioactive molecule incorporatedinto a matrix of poly-DL-lactide-co-glycolide (PLG).

Administration of the pharmaceutical composition comprising an inventivemultispecific antibody analog disclosed herein, e.g., in the form of asterile aqueous solution, may be done in a variety of ways, including,but not limited to orally, subcutaneously, intravenously, intranasally,intraotically, transdermally, topically (e.g., gels, salves, lotions,creams, etc.), intraperitoneally, intramuscularly, intrapulmonary,vaginally, parenterally, rectally, or intraocularly. In some instances,for example for the treatment of wounds, inflammation, etc., theinventive multispecific antibody analog may be directly applied as asolution or spray. As is known in the art, the pharmaceuticalcomposition may be formulated accordingly depending upon the manner ofintroduction.

Subcutaneous administration may be used in circumstances where thepatient may self-administer the pharmaceutical composition. Many proteintherapeutics are not sufficiently potent to allow for formulation of atherapeutically effective dose in the maximum acceptable volume forsubcutaneous administration. This problem may be addressed in part bythe use of protein formulations comprising arginine-HCl, histidine, andpolysorbate. Inventive multispecific antibody analogs disclosed hereinmay be more amenable to subcutaneous administration due to, for example,increased potency, improved serum half-life, or enhanced solubility. Asis known in the art, protein therapeutics are often delivered by IVinfusion or bolus. The inventive multispecific antibody analogsdisclosed herein may also be delivered using such methods. For example,administration may be by intravenous infusion with 0.9% sodium chlorideas an infusion vehicle.

Pulmonary delivery may be accomplished using an inhaler or nebulizer anda formulation comprising an aerosolizing agent. For example, AERx®inhalable technology commercially available from Aradigm, or Inhance™pulmonary delivery system commercially available from NektarTherapeutics may be used. Furthermore, inventive multispecific antibodyanalogs disclosed herein may be amenable to oral delivery.

In addition, any of a number of delivery systems are known in the artand may be used to administer the inventive multispecific antibodyanalogs disclosed herein. Examples include, but are not limited to,encapsulation in liposomes, microparticles, microspheres (e.g., PLA/PGAmicrospheres), and the like. Alternatively, an implant of a porous,non-porous, or gelatinous material, including membranes or fibers, maybe used. Sustained release systems may comprise a polymeric material ormatrix such as polyesters, hydrogels, poly(vinylalcohol), polylactides,copolymers of L-glutamic acid and ethyl-L-glutamate, ethylene-vinylacetate, lactic acid-glycolic acid copolymers such as the Lupron Depot®,and poly-D-(−)-3-hydroxyburyric acid. It is also possible to administera nucleic acid encoding an inventive multispecific antibody analogdisclosed herein, for example by retroviral infection, direct injection,or coating with lipids, cell surface receptors, or other transfectionagents. In all cases, controlled release systems may be used to releasethe inventive multispecific antibody analog at or close to the desiredlocation of action.

The dosing amounts and frequencies of administration are, in oneembodiment, selected to be therapeutically or prophylacticallyeffective. As is known in the art, adjustments for protein degradation,systemic versus localized delivery, and rate of new protease synthesis,as well as the age, body weight, general health, sex, diet, time ofadministration, drug interaction and the severity of the condition maybe necessary, and will be ascertainable with routine experimentation bythose skilled in the art.

The concentration of the therapeutically active inventive multispecificantibody analog in the formulation may vary from about 0.1 to 100 weight%. In one embodiment, the concentration of the inventive multispecificantibody analog is in the range of 0.003 to 1.0 molar. In order to treata patient, a therapeutically effective dose of the inventivemultispecific antibody analog disclosed herein may be administered. By“therapeutically effective dose” herein is meant a dose that producesthe effects for which it is administered. The exact dose will depend onthe purpose of the treatment, and will be ascertainable by one skilledin the art using known techniques. Dosages may range from 0.0001 to 100mg/kg of body weight or greater, for example 0.1, 1, 10, or 50 mg/kg ofbody weight. In one embodiment, dosages range from 1 to 10 mg/kg.

In some embodiments, only a single dose of the inventive multispecificantibody analogs is used. In other embodiments, multiple doses of theinventive multispecific antibody analog are administered. The elapsedtime between administrations may be less than 1 hour, about 1 hour,about 1-2 hours, about 2-3 hours, about 3-4 hours, about 6 hours, about12 hours, about 24 hours, about 48 hours, about 2-4 days, about 4-6days, about 1 week, about 2 weeks, or more than 2 weeks.

In other embodiments the inventive multispecific antibody analogsdisclosed herein are administered in metronomic dosing regimes, eitherby continuous infusion or frequent administration without extended restperiods. Such metronomic administration may involve dosing at constantintervals without rest periods. Typically such regimens encompasschronic low-dose or continuous infusion for an extended period of time,for example 1-2 days, 1-2 weeks, 1-2 months, or up to 6 months or more.The use of lower doses may minimize side effects and the need for restperiods.

In certain embodiments the inventive multispecific antibody analogsdisclosed herein and one or more other prophylactic or therapeuticagents are cyclically administered to the patient. Cycling therapyinvolves administration of a first agent at one time, a second agent ata second time, optionally additional agents at additional times,optionally a rest period, and then repeating this sequence ofadministration one or more times. The number of cycles is typically from2-10. Cycling therapy may reduce the development of resistance to one ormore agents, may minimize side effects, or may improve treatmentefficacy.

The inventive multispecific antibody analogs disclosed herein may beadministered concomitantly with one or more other therapeutic regimensor agents. The additional therapeutic regimes or agents may be used toimprove the efficacy or safety of the inventive multispecific antibodyanalog. Also, the additional therapeutic regimes or agents may be usedto treat the same disease or a comorbidity rather than to alter theaction of the inventive multispecific antibody analog. For example, aninventive multispecific antibody analog disclosed herein may beadministered to the patient along with chemotherapy, radiation therapy,or both chemotherapy and radiation therapy.

The terms “in combination with” and “co-administration” are not limitedto the administration of said prophylactic or therapeutic agents atexactly the same time. Instead, it is meant that the inventivemultispecific antibody analog disclosed herein and the other agent oragents are administered in a sequence and within a time interval suchthat they may act together to provide a benefit that is increased versustreatment with only either the inventive multispecific antibody analogdisclosed herein or the other agent or agents. In some embodiments,inventive multispecific antibody analogs disclosed herein and the otheragent or agents act additively, and sometimes synergistically. Suchmolecules are suitably present in combination in amounts that areeffective for the purpose intended. The skilled medical practitioner candetermine empirically, or by considering the pharmacokinetics and modesof action of the agents, the appropriate dose or doses of eachtherapeutic agent, as well as the appropriate timings and methods ofadministration.

The inventive multispecific antibody analogs disclosed herein may beadministered in combination with one or more other prophylactic ortherapeutic agents, including but not limited to cytotoxic agents,chemotherapeutic agents, antibiotics, antifungal agents, antiviralagents, cytokines, growth inhibitory agents, anti-hormonal agents,kinase inhibitors, anti-angiogenic agents, cardioprotectants,immunostimulatory agents, immunosuppressive agents, agents that promoteproliferation of hematological cells, angiogenesis inhibitors, proteintyrosine kinase (PTK) inhibitors, other antibodies, Fc fusions, orimmunoglobulins, or other therapeutic agents. The therapies of theinvention may be combined with other immunotherapies. The therapies ofthe invention may be combined with antagonists of chemokines orcytokines, including but not limited to antibodies and Fc fusions.

The inventive multispecific antibody analogs disclosed herein may becombined with other therapeutic regimens. For example, in oneembodiment, the patient to be treated with an inventive multispecificantibody analog disclosed herein may also receive radiation therapy.Radiation therapy can be administered according to protocols commonlyemployed in the art and known to the skilled artisan. Such therapyincludes but is not limited to cesium, iridium, iodine, or cobaltradiation. The radiation therapy may be whole body irradiation, or maybe directed locally to a specific site or tissue in or on the body, suchas the lung, bladder, or prostate. Optionally, the radiation therapy maybe administered as a single dose or as multiple, sequential doses. Theskilled medical practitioner can determine empirically the appropriatedose or doses of radiation therapy useful herein. In accordance withanother, an inventive multispecific antibody analog disclosed herein andone or more other anti-cancer therapies are employed to treat cancercells ex vivo. It is contemplated that such ex vivo treatment may beuseful in bone marrow transplantation and particularly, autologous bonemarrow transplantation. For instance, treatment of cells or tissue(s)containing cancer cells with an inventive multispecific antibody analogand one or more other anti-cancer therapies, such as described above,can be employed to deplete or substantially deplete the cancer cellsprior to transplantation in a recipient patient. It is of coursecontemplated that the inventive multispecific antibody analogs disclosedherein may employ in combination with still other therapeutic techniquessuch as surgery.

EXAMPLES Example 1 Enrichment of Antibody Population from NaïveLibraries Against Human Epidermal Growth Factor Receptor 2 (HER2) andHuman Epidermal Growth Factor Receptor 3 (HER3)

Eight naïve human synthetic yeast libraries each of ˜10⁹ diversity(˜10¹⁰ total diversity of all libraries combined) were prepared andpropagated in selective media. Design and generation of libraries,transformation of such libraries into host cells, for example yeastcells, MACS and FACS selections and reagents for performing such, andthe like are described in, for example, WO2009036379, WO2010105256, andWO2012009568.

For discovery of antibodies having specificity for HER2 yeast cells(˜10¹⁰ cells/library) were incubated with 3 ml of 20 nM b-HER2-Fc or 200nM biotinylated HER2 (B-HER2) for 20 min at room temperature in PBSF(phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin(BSA)) and magnetic bead assisted cell sorting (MACS) was employedutilizing the Miltenyi MACs system (Siegel et al., 2004) for two roundsof enrichment.

For HER3 IgG selections, eight naïve libraries were pulled together toyield two master libraries of four naïve libraries each. Yeast cells(˜10¹⁰ cells/library) were incubated with 3 ml of 25 nM biotinylatedHER3 (b-HER3) or 10 nM b-HER3-Fc were incubated for 20 min at roomtemperature in PBSF.

Following incubation with antigen (either HER2 or HER3) cells werewashed with 50 ml ice-cold wash buffer, and cell pellets werere-suspended in 40 ml wash buffer. Approximately 500 μl streptavidinMicroBeads were added to the re-suspended yeast and subsequentlyincubated for 15 min at 4° C. Yeast cells were washed and resuspended in5 ml PBSF buffer, and loaded onto a Miltenyi LS column. The column waswashed 3 times with 3 ml PBSF and then removed from the magnetic field,and the yeast cell of interest were eluted with 5 ml of growth media andthen grown overnight. The heavy chains of this enriched populationserved as the input for generation of restricted libraries that furtherincluded predetermined light chains (See, e.g., FIGS. 1, 3, 4 and BriefDescription of these Figures).

Extraction of Plasmid DNA Encoding Heavy and Light Chain from theEnriched HER2 and HER3 Binding Population

After 2 rounds of magnetic activated cell sorting (MACS), enriched HER2and HER3 binding yeast population were independently subjected to thesmash and grab procedure using Zymoprep™ II Yeast Plasmid Miniprep Kit(Zymo Research). DNA extraction was performed with slight modificationfrom manufacturer's protocol. Briefly, 0.5-1 OD of freshly grown yeastwere incubated with 6 μL of Zymolase in 200 μL solution 1 and incubatedin a shaker at 37° C. for an hour. Following zymolase treatment, 200 μLof solution 2 was added and mixed thoroughly to lyse the cells and thenimmediately added 400 μL of solution 3 to neutralize the lysate mixture.Cell lysate was discarded by spinning the centrifuge tube for 10 mins at21K g on a table top centrifuge (Eppendorf Centrifuge 5224). Thesupernatant was transferred to a zymo spin-I column and spin at 10K gfor 30 seconds. 550 μL was added to the column and spin down for 60seconds. DNA was eluted with either 100 μL of water or TE buffer.

Amplification of Heavy Chain and Light Chain Plasmid in E. coli

Approximately 3 μL of DNA extracted from smash and grab procedure waselectroporated into 25 μL of E. coli (NEB Turbo ElectrocompetentC2986K). The cell/DNA mix was transferred into a chilled 2 mm cuvetteand electroporated under the following conditions, voltage: 2100 V,capacitance: 25 μF, resistance: 100 S2. Immediately, followingelectroporation, cells were rescued by adding 970 μL of SOC media to thecuvette and transferred to a 15 ml culture tube followed by incubationfor 45 minutes with shaking at 37° C. Cells were then pelleted bycentrifuging at 2400 g and re-suspended in 10 ml LB media+50 μG/mlcarbenicillin and grown overnight. For each sample 2 minipreps wereperformed using standard Qiagen miniprep kit. Typical yield from theminipreps were in the range of 10-15 μg of DNA.

Example 2 Generation of HC Chain Dominant Libraries with FivePreselected Light Chains from EGFR Binders

Plasmid DNA obtained from the E. coli. minipreps obtained in Example 1was subjected to digestion with restriction enzymes Nco1-HF and Sbf1-HF(unique restriction sites in light chain (LC) plasmid) and HindIII-HF(unique restriction sites in heavy chain (VH) plasmid). Briefly, around8-10 μg of DNA was digested in cut smart buffer with 200 units ofNco1-HF, Sbf1-HF and HindIII-HF for 4 hours at 37° C. Digested DNA wascombined with a PCR amplified DNA fragment that has homology to aportion of the HC plasmid that coincides with the intended recombinationsite, such that the VH will recombine in-frame with the remainder of thecoding region of full heavy chain (VH+hinge+Fc) and thus give rise to anopen reading frame that affords expression of the full length IgG heavychain. This DNA mixture was electroporated into yeast containing thedesired light chain plasmids (5 light chains obtained from 5EGFR-binding IgGs). HC plasmid was repaired in yeast via homologousrecombination. The electroporated yeast library with HC diversities andfixed light chains were grown in selective media. Using this procedure,4 libraries for HER2 and 2 libraries for HER3 were generated. Therealized diversity of the libraries were in the range of 10⁶ to 10⁷.

Example 3 Discovery of HER2 and HER3 IgGs from HC Dominant (Biased)Libraries Paired with 5 Predefined Light Chains from EGFR BindersIdentification of Parent IgGs Having Common Light Chain

Magnetic bead sorting technique utilizing the Miltenyi MACs system wasperformed (Siegel et al., 2004) for the first round of selections (see,e.g., FIGS. 3 and 11).

Briefly, 1.5 ml of 20 nM b-HER2-Fc or 20 nM b-HER3-Fc were incubatedwith restricted (also known as biased) yeast libraries (as generatedabove) for 20 min at room temperature in PBSF. Magnetic activated cellsorting (MACS) was carried out as described above. Following the firstround of MACS selection, three rounds of Fluorescence Activated CellSorting (FACS) were performed. For FACS selection, libraries wereincubated with decreasing concentrations of antigens (5 nM b-HER2-HIS or10 nM b-HER3-HIS) and subsequently stained with LC-FITC (diluted 1:100)and either SA-633 (diluted 1:500) or EA-PE (diluted 1:50) for 15 min at4° C. Cells were washed twice and re-suspended in 0.4 ml wash buffer andtransferred to strainer-capped sort tubes. Sorting was performed using aFACS ARIA sorter (BD Biosciences) and sort gates were determined toselect for high affinity binders with good expression. After the finalround of sorting, yeast were plated and individual colonies were pickedfor characterization.

Affinity Maturation by Introducing CDRH1 and CDRH2 Diversities intoNaive HER2 and HER3 Parent IgGs Having Common Light Chain

A pool of gapped HC vector with diversity in CDRH1 and CDRH2 wasco-transformed with the CDRH3s obtained from the parent IgGs isolated asdescribed above into a yeast strain containing a light chain plasmidharboring the light chain from the parent IgG. The result was acombinatorial library containing the same LC and CDRH3 from each parentIgG, but with diversity in CDRH1, CDRH2 or CDRH1 and CDRH2. Thetheoretical diversity of this newly built library was in the range of10⁸. The libraries were propagated in selective media and weresubsequently subjected to MACS and FACS selections.

HER2 IgG optimization: For the 1^(st) round of selection libraries wereincubated with 1.5 ml of 20 nM b-HER2-Fc in PBSF for 20 mins at roomtemperature and subsequently performed MACS selection as mentionedabove. For later rounds of selection affinity pressure was applied bycompeting off IgG bound biotinylated antigen with excess of unlabeledfree antigen for appropriate amount of time and subsequently FACSsorting the best affinity yeast clones with high expression. After thefinal round of sorting, yeast were plated and individual colonies werepicked for characterization.

HER3 IgG optimization: For the 1^(st) round of selection libraries wereincubated with 1.5 ml of 25 nM b-HER3-HIS in PBSF for 20 mins at roomtemperature and subsequently followed MACS selection as mentioned above.For later rounds of selection affinity pressure was applied by competingoff IgG bound biotinylated antigen with excess of unlabeled free antigenfor appropriate amount of time and subsequently FACS sorting the bestaffinity yeast clones with high expression. After the final round ofsorting, yeast were plated and individual colonies were picked forcharacterization.

Selection of Improved Progenies by Introducing Stochastic Mutations inthe Variable Region of Heavy Chain (VU) of Parent HER2

Affinity maturation of the parent HER2 IgG was performed bystochastically mutagenizing the variable region (VH) by using nucleotideanalogue mutagenesis. Briefly, mutated VH segments of the HC were cotransformed with gapped HC plasmid into a yeast strain containing thelight chain plasmid of the parent HER2 IgG. The HC plasmid was repairedby homologous recombination in the yeast, such that the mutagenized VHregions recombined in-frame with gapped HC vector such that open readingframes afforded expression of full length IgGs. The theoreticaldiversity of this newly built library was in the range of 5×10⁶.

The libraries were propagated and were subsequently subjected to MACSand FACS selection. Briefly, for the 1^(st) round of selection yeastcells (˜10⁸ cells/library) were incubated with 1.5 ml of 10 b-HER2-Hisfor 20 min at room temperature in PBSF. Following antigen incubation,cells were labeled with LC-FITC and EAPE and sorted the binders on FACS.For subsequent selections, to yield the best binders either antigenconcentration was tittered down to 500 pM or K_(off) pressure wasapplied by incubating the biotinylated antigen bound library with excessof unlabeled antigen. After the final round of sorting, yeast wereplated and individual colonies were picked for characterization.

High Throughput Expression and Purification of Yeast Derived IgGs HavingCommon Light Chain

After successful completion of FACS selection sorted yeast were platedon selective media and individual colonies were picked and sequencedconfirmed. Yeast clones expressing a specific IgG were cultivated in24-well plates by means such as those available in the art (see, e.g.,WO2009036379, WO2010105256, and WO2012009568). IgGs were expressed andrecovered from the supernatant by a single step of Protein A (MabSelectSuRe, GE Healthcare) purification executed on a liquid handling robot(Biomek FX Liquid Handler).

Octet RED384 Surface Based Binding Assessments

Fortebio KD measurements were performed as previously described (see,e.g., Estep et al., MAbs, Vol. 5(2), pp. 270-278 (2013). Briefly, ligand(antibody or antigen) was loaded to the sensor followed by a shortbaseline in PBSF, the sensors were exposed to analyte at a singleconcentration in PBSF for an association step. Dissociation wasmonitored in PBSF. For dual binding assessment sensors were exposed to2^(nd) analyte instead of PBSF. ForteBio's data analysis software wasused to fit the data to a 1:1 binding model to extract an associationrate and dissociation rate. The K_(D) was calculated using the ratiok_(d)/k_(a).

Octet RED384 Epitope Binning

Epitope binning assays were performed as previously described (Estep etat 2013). Briefly, control antibodies were loaded onto AHQ sensors andremaining Fc-binding sites on the sensor were blocked with a human IgG1antibody. Sensors were exposed to antigen followed by the secondantibody. Additional binding by the second antibody indicates anunoccupied epitope (non-competitor), while no binding indicates epitopeblocking (competitor).

Cloning of IgGs and Multispecific Antibody Analogs for Expression inHuman Embryonic Kidney (HEK) Cell Line

A mammalian expression vector containing suitable unique restrictionsites and harboring the coding sequence for CH1, hinge, CH2, and CH3regions of human IgG1 was used as the template vector for all variableheavy chain (VH) related cloning. For variable light chain (VK) cloning,a vector containing suitable unique restriction sites and harboring thecoding sequence for CK region was used as the template vector.

Variable heavy chain (VH) regions of IgG1 from EGFR-binding IgGs,HER2-binding IgGs, and HER3 IgGs were independently amplified with aforward primer that contains flanking Nhe1 restriction site and areverse primer that contains flanking Xho1 site. The amplified VHregions and the vector were each digested with Nhe1 and Xho1, andsubsequently teach VH region was independently inserted into the vectorvia ligation using T4 DNA ligase.

Variable light chain (VK) regions of IgG1 from HER 2 IgGs and HER3 IgGswere independently amplified with a forward primer that containsflanking Xho1 restriction site and a reverse primer that containsflanking BSiW1 restriction site. Each amplified VK region and the vectorwere digested with Xho1 and BSiW1 subsequently the VK region wasinserted into the vector via ligation using T4 DNA ligase.

Cloning of the C-terminal Fab regions (see, e.g., FIGS. 5, 6, 7, 8, 9,12, 13, and 14 for depictions of exemplary multispecific antibodyanalogs and position of Fab regions therein) were accomplished byligation of synthetic double stranded Fab-encoding DNA to the vectorcontaining the desired full length IgG1. The Fab-encoding DNA contains aHindIII restriction site at 5′ end followed by a 15-mer ((Gly₄S)₃)linker (SEQ ID NO: 97), followed by VH region and finally the CH1 regionfollowed by a Not1 restriction site at 3′ end. Following digestion ofthe vector and the insert with HindIII and Not1, the Fab-encoding insertwas subsequently ligated to the vector.

E. coli cells were then transformed with ligated vector viaelectroporation, and grown on selective plates. Colonies were pickedfrom the plates, grown up, and plasmid DNA extracted andsequence-confirmed.

As indicated above and in the, e.g., FIGS. 7, 8, 9, 12, 13, and 14,exemplary multispecific antibody analogs prepared in accordance with theinventive methods are comprised of two copies of a first polypeptide andfour copies of a second polypeptide, wherein:

the first polypeptide comprises, in the following N-terminus toC-terminus order: N-term—Antigen 1 (e.g., EGFR) VH—CH1—Hingeregion—CH2—CH3—Antigen 2 (e.g., HER2) VH—CH1—C-term; and

the second polypeptide comprises, in the following N-terminus toC-terminus order: N-term—VL—CK—C-term;

wherein the second polypeptide comprises a common light chain that iscompatible with VH for Antigen 1 and VH for Antigen 2.

As described above and illustrated in FIGS. 7, 8, 9, 12, 13, and 14,four exemplary multispecific antibody analogs were prepared:

an N-terminal—EGFR::C-terminal HER2 multispecific antibody analog(“Analog 1”);

an N-terminal HER2::C-terminal EGFR multispecific antibody analog(“Analog 2”);

an N-terminal EGFR::C-terminal HERS multispecific antibody analog(“Analog 3”); and

an N-terminal HER3::C-terminal EGIR multispecific antibody analog(“Analog 4”).

Exemplary such first polypeptide amino acid sequences are as follows:

Analog 1: N-term-EGF::C-term-HER2 (see FIGS. 7, 8, and 9)

First polypeptide amino acid sequence (N-term—EGFR VH—CH1—hingeregion—CH2—CH3—HER2 VH—CH1—C-term):

(SEQ ID NO: 98) QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARTNLYSTPFDIWGGTNWTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGGSISSGGYYWSWIRQPPGKGLEWIGIIYYSGWTNYNPSLKSVTISVDASRNQFSLKLSSVTAADTAVYYCARGVGPDFWSGYSYSSYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSC

Second polypeptide amino acid sequence (N-term—VL—CK—C-term):

(SEQ ID NO: 79) DIQLTQSPSTLSASVGDRVTITCRASQAISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCHQYQSYSWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC

Analog 2: N-term-HER2::C-term-EGFR (see, e.g., FIGS. 7, 8, and 9)

First polypeptide amino acid sequence (N-term—HER2 VH—CH1—hingeregion—CH2—CH3—EGFR VH—CH1—C-term):

(SEQ ID NO: 76) QVQLQESGPGLVKPSETLSLTCTVSGGSISSGGYYWSWIRQPPGKGLEWIGIIYYSGWTNYNPSLKSRVTISVDASRNQFSLKLSSVTAADTAVYYCARGVGPDFWSGYSYSSYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGGSVSSGSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARTNLYSTPFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSC

Second polypeptide amino acid sequence (N-term—VL—CK—C-term):

(SEQ ID NO: 79) DIQLTQSPSTLSASVGDRVTITCRASQAISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCHQYQSYSWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC

Analog 3: N-term-EGFR::C-term-HER3 (see, e.g., FIGS. 12, 13, and 14)

First polypeptide amino acid sequence (N-term—EGFR VH—CH1—hingeregion—CH2—CH3—HER3 VH—CH1—C-term):

(SEQ ID NO: 74) QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARTNLYSTPFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSQLQLQESGPGLVKPSETLSLTCTVSGGSINSSSYYWQWIRQPPGKGLEWIGEIAYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGQQWAAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSC

Second polypeptide amino acid sequence (N-term—VL—CK—C-term):

(SEQ ID NO: 78) DIQUMSPSSVSASVGDRATITICRASQDISSWLAWYQQKPGKAPKLUYSSLQSGVPSRFSGSGSGTDFTLTNSLQPEDFATYYCQQEHDFPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC

Analog 4: N-term-HER3::C-term-EGFR (see, e.g., FIGS. 12, 13, and 14)

First polypeptide amino acid sequence (N-term—HER3 VH—CH1—hingeregion—CH2—CH3—EGFR VH—CH1—C-term):

(SEQ ID NO: 77) QLQLQESGPGLVKPSETLSLTCTVSGGSINSSSYYWQWIRQPPGKGLEWIGEIAYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGQQWAAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGGSVSSGSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARTNLYSTPFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSC

Second polypeptide amino acid sequence (N-term—VL—CK—C-term):

(SEQ ID NO: 78) DIQLTQSPSSVSASVGDRVTITCRASQDISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQEHDFPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC

Mammalian Expression and Purification of IgGs and Multispecific AntibodyAnalogs

Following 7 days of HEK transfection with appropriate vectors, IgGs ormultispecific antibody analogs as described above and as depicted in theFigures were harvested and purified by a single step of protein Apurification. Protein A resin (MabSelect SuRe (GE Healthcare) resin wasfirst equilibrated in wash buffer (phosphate buffered saline, pH 7.4)and then IgG or bispecific samples were applied to the column. Sampleswere eluted from the column by the addition of elution buffer (200 mMAcetic Acid, pH 2.0). Once eluted, the samples were neutralized with 2 MHEPES, pH 8.0.

Cell Labeling IgGs and Multispecific Antibody Analogs

IgG and multispecific antibody analogs were incubated withEGFR-overexpressing A431 cells, HER2-overexpressing BT474 cells, andHER3-overexpressing MDA-MB-453 cells. CHO-S cells were used as anegative control.

Around 5×10⁴ cells were incubated with 100 μL of 100 nM of either IgG ormultispecific antibody analogs for 30 mins at 4° C. Cells weresubsequently washed and stained with goat anti-human IgG-RPE for 15 minsat 4° C. Samples were run on BD FACS Canto II instrument.

MSD-SET K_(D) Measurements

Equilibrium affinity measurements performed as previously described(Estep et al., 2013). Solution equilibrium titrations (SET) wereperformed in PBS+0.1% IgG-Free BSA (PBSF) with antigen held constant at50 pM and incubated with 3-to 5-fold serial dilutions of antibodystarting at around 50 nM. Antibodies (20 nM in PBS) were coated ontostandard bind MSD-ECL plates overnight at 4° C. or at room temperaturefor 30 min. Plates were then blocked for 30 min with shaking at 700 rpm,followed by three washes with wash buffer (PBSF+0.05% Tween 20). SETsamples were applied and incubated on the plates for 150s with shakingat 700 rpm followed by one wash. Antigen captured on a plate wasdetected with 250 ng/ml sulfotag-labeled streptavidin in PBSF byincubation on the plate for 3 min. The plates were washed three timeswith wash buffer and then read on the MSD Sector Imager 2400 instrumentusing 1× Read Buffer T with surfactant. The percent free antigen wasplotted as a function of titrated antibody in Prism and fit to aquadratic equation to extract the K_(D). To improve throughput, liquidhandling robots were used throughout MSD-SET experiments, including SETsample preparation.

Melting Temperature (Tm) Measurement by Differential ScanningFluorometry (DSP)

Briefly, 10 μL of 20× sypro orange dye was added to 20 μL of 0.5 mg/mlFab, IgG or bispecific solution and were mixed thoroughly. DSF Tm wereperformed on a BioRad CFX96 RT PCR instrument by ramping temperature at0.5° C. increment from 40 to 95° C. At each temperature point it wasallowed to equilibrate for 2 mins. The melting point of the protein wasobtained as the lowest point of first derivative plot, as calculated bythe software included with the RT-PCR machine.

Size Exclusion Chromatography (SEC)

SEC chromatogram were acquired by running samples through a TSKgel SuperSW3000 column (Tosoh Bioscience LLC) on an Agilent 1100 HPLC system.Briefly, 5 μG of samples were injected with PBS as running buffer at aflow rate of 0.4 ml/min.

The results, collectively depicted in, e.g., FIGS. 4 and 7, (for EGFRand HER2 IgGs having common light chains) and FIGS. 11 and 12 (for EGFRand HER3 IgG having common light chains, demonstrate that the methodsgenerating restricted libraries (also known as biased libraries andenriched libraries) as described above, which include: isolating lightchains from IgGs identified from selections using naïve librariesinterrogated with a first antigen (e.g., EGFR); isolating heavy chainsfrom IgGs isolated from selections using naïve libraries interrogatedwith a second antigen (e.g., HER2 or HER3); combining the light chainsand heavy chains to generate the restricted library; afford librariesfrom which IgGs can be identified having specificity to both antigen 1and antigen 2 (e.g., EGFR and, respectively, HER2 or HER3), and whichshare a common light chain.

The results also demonstrate that when the antigen binding regions arereformatted as IgG-Fab multispecific antibody analogs having commonlight chains, either as: EGFR-HER2; EGFR-HER2; HER2-EGFR; or HER3-EGFR;IgG-Fabs, such analogs retain specificity for the respective antigenwith affinities comparable, or even superior to, the affinities observedfor the antigen binding regions in the context of IgGs (See, e.g., FIGS.7 and 12).

Such multispecific antibody analogs were also observed to elute from SECcolumns as largely monomeric species at percentages comparable to thatobserved for corresponding IgGs (see, e.g., FIGS. 8 and 13).Additionally, such multispecific antibody analogs were observed topossess melting temperatures comparable to those observed forcorresponding IgGs (see, e.g., FIGS. 9 and 14). Accordingly, suchmultispecific antibody analogs having common light chains demonstratefavorable stability and conformation profiles comparable to full lengthIgGs.

Additional exemplary Embodiments of certain aspects of the invention areas follows:

Embodiment 1

A method of making a multispecific antibody analog comprising at leasttwo first antigen binding regions and at least two second antigenbinding regions, said first and second antigen binding regions having acommon light chain, wherein first antigen binding regions have adifferent antigen specificity than the second antigen binding regions,the method comprising:

i) obtaining at least one light chain from a first antigen bindingregion having specificity for the first antigen, wherein the firstantigen binding region comprises said at least one light chain and aheavy chain;

ii) obtaining heavy chains from the output of a selection performed froma naïve library against a second antigen;

iii) preparing a restricted library comprising heavy chains obtained instep ii) and the at least one light chain obtained in step i);

iv) performing a second selection against the second antigen from thelibrary prepared in step iii);

v) obtaining an multispecific antibody comprising the second antigenbinding region from the selection performed in step iv), wherein thesecond antigen binding region comprises the at least one light chainobtained in step i);

vi) incorporating the first antigen binding region and the secondantigen binding region into a multispecific multispecific antibodyformat, wherein the format comprises: an IgG moiety comprising either:

-   -   a) the first antigen binding region; or    -   b) the second antigen binding region; and        two Fab moieties, wherein each Fab moiety comprises either:    -   a) the second antigen binding region; or    -   b) the first antigen binding region;        wherein the N-terminus of the heavy chain of one Fab moiety is        linked to the C-terminus of the Fc region of one heavy chain of        the IgG moiety via a linker moiety, and the N-terminus of the        heavy chain of the other Fab moiety is linked to the C-terminus        of the Fc region of the other heavy chain of the IgG moiety via        a linker moiety;        thereby generating the multispecific multispecific antibody        analog.

Embodiment 2

The method according to Embodiment 1, wherein each linker moietyindependently comprises a peptide from 1 to 75 amino acids in length,inclusive.

Embodiment 3

The method according to any one of Embodiments 1 and 2, wherein one ormore of the linker moieties independently comprises at least one of the20 naturally occurring amino acids.

Embodiment 4

The method according to any one of Embodiments 1 through 3, wherein theone or more of the linker moieties independently comprises at least onenon-natural amino acid incorporated by chemical synthesis,post-translational chemical modification or by in vivo incorporation byrecombinant expression in a host cell.

Embodiment 5

The method according to any one Embodiments of 1 through 4, wherein theone or more of the linker moieties independently comprises one or moreamino acids selected from the group consisting of serine, glycine,alanine, proline, asparagine, glutamine, glutamate, aspartate, andlysine.

Embodiment 6

The method according to any one of Embodiments 1 through 5, wherein theone or more of the linker moieties independently comprises a majority ofamino acids that are sterically unhindered.

Embodiment 7

The method according to any one of Embodiments 1 through 6, wherein theone or more of the linker moieties independently comprises one or moreof the following: an acidic linker, a basic linker, and a structuralmotif.

Embodiment 8

The method according to any one of Embodiments 1 through 7, wherein oneor more of the linker moieties independently comprises: polyglycine,polyalanine, poly(Gly-Ala), or poly(Gly-Ser).

Embodiment 9

The method according to any one of Embodiments 1 through 8, wherein oneor more of the linker moieties independently comprises: a polyglycineselected from the group consisting of: (Gly)3 (SEQ ID NO: 1), (Gly)4(SEQ ID NO: 2), and (Gly)5 (SEQ ID NO: 3).

Embodiment 10

The method according to any one of Embodiments 1 through 9 wherein oneor more of the linker moieties independently comprises (Gly)₃Lys(Gly)₄(SEQ ID NO: 4); (Gly)₃AsnGlySer(Gly)₂ (SEQ ID NO: 5); (Gly)₃Cys(Gly)₄(SEQ ID NO: 6); and GlyProAsnGlyGly (SEQ ID NO: 7).

Embodiment 11

The method according to any one of Embodiments 1 through 10, wherein oneor more of the linker moieties independently comprises a combination ofGly and Ala.

Embodiment 12

The method according to any one of Embodiments 1 through 11, wherein oneor more of the linker moieties independently comprises a combination ofGly and Ser.

Embodiment 13

The method according to any one of Embodiments 1 through 12, wherein oneor more of the linker moieties independently comprises a combination of:

Gly and Glu; or

Gly and Asp.

Embodiment 14

The method according to any one of Embodiments 1 through 13, wherein oneor more of the linker moieties independently comprises a combination ofGly and Lys.

Embodiment 15

The method according to any one of Embodiments 1 through 14, wherein oneor more of the linker moieties independently comprises a sequenceselected from group consisting of: [Gly-Ser]_(n) (SEQ ID NO: 8);[Gly-Gly-Ser]_(n) (SEQ ID NO: 9); [Gly-Gly-Gly-Ser]_(n) (SEQ ID NO: 10);[Gly-Gly-Gly-Gly-Ser]_(n) (SEQ ID NO: 11);[Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly]_(n) (SEQ ID NO: 12);[Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly]_(n) (SEQ IDNO: 13); [Gly-Gly-Gly-Gly-SerGly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly]_(n) (SEQ ID NO:14);[Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly]_(n)(SEQ ID NO: 15);[Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly]_(n)(SEQ ID NO: 16);[Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly]_(n)(SEQ ID NO: 17); and combinations thereof; where n is an integerselected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, and 75.

Embodiment 16

The method according to any one of Embodiments 1 through 15, wherein oneor more of the linker moieties independently comprises a sequenceselected from the group consisting of: [Gly-Glu]_(n) (SEQ ID NO: 18);[Gly-Gly-Glu]_(n) (SEQ ID NO: 19); [Gly-Gly-Gly-Glu]_(n) (SEQ ID NO:20); [Gly-Gly-Gly-Gly-Glu]_(n) (SEQ ID NO: 21); [Gly-Asp]n (SEQ ID NO:22); [Gly-Gly-Asp]_(n) (SEQ ID NO: 23); [Gly-Gly-Gly-Asp]_(n) (SEQ IDNO: 24); [Gly-Gly-Gly-Gly-Asp]_(n) (SEQ ID NO: 25); and combinationsthereof; where n is an integer selected from the group consisting of 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, and75.

Embodiment 17

The method according to any one of Embodiments 1 through 16, wherein atleast one of the first and second antigen binding regions comprises atleast one humanized variable heavy domain or at least one humanizedvariable light domain.

Embodiment 18

The method according to any one of Embodiments 1 through 17, wherein atleast one of the first and second antigen binding regions comprises atleast one complimentary determining region CDR that is derived from anon-human multispecific antibody or multispecific antibody fragment.

Embodiment 19

The method according to any one of Embodiments 1 through 18, wherein atleast one of the first and second antigen binding regions binds anepitope from a tumor associated antigen, a hormone receptor, a cytokinereceptor, chemokine receptor, a growth factor receptor, an immuneactivating receptor, a hormone, a cytokine, a chemokine, a growthfactor, a G protein-coupled receptor, or a transmembrane receptor.

Embodiment 20

The method according to any one of Embodiments 1 through 19, wherein atleast one of the first and second antigen binding regions binds a targetassociated with an autoimmune disorder, an inflammatory disorder, anoncological disorder, neuromuscular disorder, a neurodegenerativedisorder, a metabolic disorder, or an infectious disease.

Embodiment 21

The method according to any one of Embodiments 1 through 20, wherein themultispecific antibody analog binds at least two different targets.

Embodiment 22

The method according to any one of Embodiments 1 through 21, wherein themultispecific analog binds at least three different targets.

Embodiment 23

The method according to any one of Embodiments 1 through 22, wherein themultispecific antibody analog binds at least four different targets.

Embodiment 24

The method according to any one of Embodiments 1 through 23, wherein themultispecific antibody analog binds at least one target monovalently.

Embodiment 25

The method according to any one of Embodiments 1 through 24, wherein themultispecific antibody analog binds at least two targets monovalently.

Embodiment 26

The multivalent multispecific antibody analog according to any one ofEmbodiments 1 through 72, wherein the multispecific antibody analogbinds at least three targets monovalently.

Embodiment 27

The method according to any one of Embodiments 1 through 26, wherein themultispecific antibody analog binds at least four targets monovalently.

Embodiment 28

The method according to any one of Embodiments 1 through 27, wherein atleast one of the antigen binding regions comprises or is derived from anon-human species.

Embodiment 29

The method according to any one of Embodiments 1 through 28, wherein atleast one of the antigen binding sites comprises a humanized variabledomain or a humanized CDR.

Embodiment 30

The method according to any one of Embodiments 1 through 29, wherein atleast one VH comprises a VH CDR1, a VH CDR2, and a VH CDR3 eachindependently selected from the following:

a VH CDR1 amino acid sequence selected from the group consisting of:

(SEQ ID NO: 26) GSVSSGSYYWS; (SEQ ID NO: 27) GSISSGGYYWS;(SEQ ID NO: 28) GSINSSSYYWQ; (SEQ ID NO: 29) FTLSGDWIH; (SEQ ID NO: 30)FNIKDTYIH; (SEQ ID NO: 31) FSLTNYGVH; (SEQ ID NO: 32) GSISSGGDYWQ;

a VH CDR2 amino acid sequence selected from the group consisting of:

(SEQ ID NO: 33) YIYYSGSTNYNPSLKS; (SEQ ID NO: 34) IIYYSGWTNYNPSLKS;(SEQ ID NO: 35) EIAYSGSTYYNPSLKS; (SEQ ID NO: 36) EISAAGGYTDYADSVKG;(SEQ ID NO: 37) RIYPTNGYTRYADSVKG; (SEQ ID NO: 38) VIWSGGNTDYNTPFTSR;and

ARTNLYSTPFDI; (SEQ ID NO: 39) ARGVGPDFWSGYSYSSYFDL; (SEQ ID NO: 40)ARGQQWAAFDI; (SEQ ID NO: 41) ARESRVSFEAAMDY; (SEQ ID NO: 42)SRWGGDGFYAMDY; (SEQ ID NO: 43) RALTYYDYEFAYW. (SEQ ID NO: 44)

a VH CDR3 selected from the group consisting of:

Embodiment 31

The method according to any one of Embodiments 1 through 30, wherein atleast one VL comprises a VL CDR1, a VL CDR2, and a VL CDR3 eachindependently selected from the following:

a VL CDR1 amino acid sequence selected from the group consisting of:

(SEQ ID NO: 45) RASQDISSWLA; (SEQ ID NO: 46) RASQAISSWLA;(SEQ ID NO: 47) RASQNIATDVA; (SEQ ID NO: 48) RASQDVNTAVA;(SEQ ID NO: 49) RASQSIGTNIH;

a VL CDR2 amino acid sequence selected from the group consisting of:

(SEQ ID NO: 50) AASSLQS; (SEQ ID NO: 51) DASSLES; (SEQ ID NO: 52)AASSLQS; (SEQ ID NO: 53) SASFLYS;

YASESIS (SEQ ID NO: 54); and

a VL CDR3 amino acid sequence selected from the group consisting of:

(SEQ ID NO: 55) QQEHDFPWT; (SEQ ID NO: 56) HQYQSYSWT; (SEQ ID NO: 57)QQEHDFPWT; (SEQ ID NO: 58) QQSEPEPYT; (SEQ ID NO: 59) QQHYTTPPT;(SEQ ID NO: 60) QQNNNWPTT.

Embodiment 32

The method according to any one of Embodiments 1 through 31, wherein themultispecific antibody analog comprises at least one heavy chainframework region that corresponds to or is derived from VH1-46, VH3-23,VH4-39, or VH4-61, and wherein at least one light chain framework regionthat corresponds to or is derived from VK1-05, VK1-12, or VK3-11.

Embodiment 33

The method according to any one of Embodiments 1 through 32, wherein themultispecific antibody analog comprises a VH region that comprises anamino acid sequence selected from the group consisting of:

(SEQ ID NO: 99) QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCART NLYSTPFDIWGQGTMVTVSS;(SEQ ID NO: 62) QVQLQESGPGLVKPSETLSLTCTVSGGSISSGGYYWSWIRQPPGKGLEWIGIIYYSGWTNYNPSLKSRVTISVDASRNQFSLKLSSVTAADTAVYYCARGVGPDFWSGYSYSSYFDLWGRGTLVTVSS; (SEQ ID NO: 63)QLQLQESGPGLVKPSETLSLTCTVSGGSINSSSYYWQWIRQPPGKGLEWIGEIAYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARG QQWAAFDIWGQGTMVTVSS;(SEQ ID NO: 64) EVQLVESGGGLVQPGGSLRLSCAASGFTLSGDWIHWVRQAPGKGLEWVGEISAAGGYTDYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARESRVSFEAAMDYWGQGTLVTVSS; (SEQ ID NO: 65)EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG GDGFYAMDYWGQGTLVTVSS;(SEQ ID NO: 66) QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSDDTAIYYCARALT YYDYEFAYWGQGTLVTVSS;and (SEQ ID NO: 67) QLQLQESGPGLVKPSETLSLTCTVSGGSISSGGDYWQWIRQPPGKGLEWIGEIAYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARG QQWAAFDIWGQGTMVTVSS.

Embodiment 34

The method according to any one of Embodiments 1 through 33, wherein themultispecific antibody analog comprises a VL region that comprises anamino acid sequence selected from the group consisting of:

(SEQ ID NO: 68) DIQLTQSPSSVSASVGDRVTITCRASQDISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQEHDFPWTFGG GTKVEIK;(SEQ ID NO: 69) DIQLTQSPSTLSASVGDRVTITCRASQAISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCHQYQSYSWTFGG GTKVEIK;(SEQ ID NO: 70) DIQLTQSPSSVSASVGDRVTITCRASQDISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQEHDFPWTFGG GTKVEIK;(SEQ ID NO: 71) DIQMTQSPSSLSASVGDRVTITCRASQNIATDVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSEPEPYTFGQ GTKVEIK;(SEQ ID NO: 72) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ GTKVEIK; and(SEQ ID NO: 73) DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTHGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGA GTKLELK.

Embodiment 35

The method according to any one of Embodiments 1 through 34, wherein themultispecific antibody analog comprises a polypeptide comprising, fromN-terminus to C-terminus, a first VH region, a CH1, a hinge region, aCH2 region, a CH3 region, a second VH region, and a CH1 region, theamino acid sequence of which comprises an amino acid sequence selectedfrom the group consisting of:

(SEQ ID NO: 74) QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARTNLYSTPFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSQLQLQESGPGLVKPSETLSLTCTVSGGSINSSSYYWQWIRQPPGKGLEWIGEIAYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGQQWAAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC; (SEQ ID NO: 75)QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARTNLYSTPFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGGSISSGGYYWSWIRQPPGKGLEWIGIIYYSGWTNYNPSLKSRVTISVDASRNQFSLKLSSVTAADTAVYYCARGVGPDFWSGYSYSSYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC; (SEQ ID NO: 76)QVQLQESGPGLVKPSETLSLTCTVSGGSISSGGYYWSWIRQPPGKGLEWIGIIYYSGWTNYNPSLKSRVTISVDASRNQFSLKLSSVTAADTAVYYCARGVGPDFWSGYSYSSYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGGSVSSGSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARTNLYSTPFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC; (SEQ ID NO: 77)QLQLQESGPGLVKPSETLSLTCTVSGGSINSSSYYWQWIRQPPGKGLEWIGEIAYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGQQWAAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGGSVSSGSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARTNLYSTPFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC.

Embodiment 36

The method according to any one of Embodiments 1 through 35, wherein themultispecific antibody analog comprises four copies of a polypeptidecomprising, from N-terminus to C-terminus, a VL region, and a CK region,and wherein said polypeptide heterodimerizes with compatible VH regionsof the multispecific antibody analog, the amino acid sequence of whichcomprises an amino acid sequence selected from the group consisting of:

(SEQ ID NO: 78) DIQLTQSPSSVSASVGDRVTITCRASQDISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQEHDFPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC;(SEQ ID NO: 79) DIQLTQSPSTLSASVGDRVTITCRASQAISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCHQYQSYSWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC;(SEQ ID NO: 80) DIQMTQSPSSLSASVGDRVTITCRASQNIATDVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSEPEPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC;(SEQ ID NO: 81) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC; and(SEQ ID NO: 82) DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTHGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC.

Embodiment 37

The method according to any one of Embodiments 1 through 36 wherein themultispecific antibody analog has binding specificity for an oncologytarget.

Embodiment 38

The method according to any one of Embodiments 1 through 37, wherein themultispecific antibody analog has binding specificity for one or moretargets selected from the group consisting of: EGFR, HER2, and HER3.

Embodiment 39

The method according to any one of Embodiments 1 through 38, wherein themultispecific antibody analog has binding specificity for EGFR and HER2.

Embodiment 40

The method according to any one of Embodiments 1 through 39, wherein thepolypeptide multispecific antibody analog has binding specificity forEGFR and HER3.

Embodiment 41

The method according to any one of Embodiments 1 through 40, wherein themultispecific antibody analog has binding specificity for EGFR, HER2,and HER3.

Embodiment 42

The method according to any one of Embodiments 1 through 41, wherein themultispecific antibody analog is selected from the group consisting ofthe multispecific antibody analogs described in the Examples.

Embodiment 43

The method according to any one of Embodiments 1 through 42, wherein themultispecific antibody analog is expressed by a prokaryotic host cell ora eukaryotic host cell.

Embodiment 44

The method according to any one of Embodiments 1 through 43, wherein themultispecific antibody analog is expressed by a eukaryotic host cell.

Embodiment 45

The method according to any one of Embodiments 1 through 44, wherein themultispecific antibody analog is expressed by a eukaryotic host cellselected from the group consisting of: yeast cells; Saccharomycescerevisiae cells; Pichia cells; mammalian cells; Chinese hamster ovary(CHO) cells; human embryonic kidney (HEK) cells; insect cells; Sf9cells; and Sf21 cells.

Embodiment 46

A multispecific antibody analog prepared by performing a methodaccording to any one of Embodiments 1 through 44.

Embodiment 47

A multispecific antibody analog comprising at least two first antigenbinding regions and at least two second antigen binding regions, saidfirst and second antigen binding regions having a common light chain,wherein first antigen binding regions have a different antigenspecificity than the second antigen binding regions.

Embodiment 48

A multispecific antibody analog comprising at least two first antigenbinding regions and at least two second antigen binding regions, saidfirst and second antigen binding regions having a common light chain,wherein first antigen binding regions have a different antigenspecificity than the second antigen binding regions, wherein the analogprepared by a method comprising:

i) obtaining at least one light chain from a first antigen bindingregion having specificity for the first antigen, wherein the firstantigen binding region comprises said at least one light chain and aheavy chain;

ii) obtaining heavy chains from the output of a selection performed froma naïve library against a second antigen;

iii) preparing an multispecific antibody library comprising heavy chainsobtained in step ii) and the at least one light chain obtained in stepi);

iv) performing a second selection against the second antigen from thelibrary prepared in step iii);

v) obtaining an multispecific antibody comprising the second antigenbinding region from the selection performed in step iv);

vi) incorporating the first antigen binding region and the secondantigen binding region into a multispecific antibody format, wherein theformat comprises: an IgG moiety comprising either:

-   -   a) the first antigen binding region; or    -   b) the second antigen binding region; and        two Fab moieties, wherein each Fab moiety comprises either:    -   a) the second antigen binding region; or    -   b) the first antigen binding region;        wherein the N-terminus of the heavy chain of one Fab moiety is        linked to the C-terminus of the Fc region of one heavy chain of        the IgG moiety via a linker moiety, and the N-terminus of the        heavy chain of the other Fab moiety is linked to the C-terminus        of the Fc region of the other heavy chain of the IgG moiety via        a linker moiety;        thereby generating the multispecific antibody analog.

Embodiment 49

The multispecific antibody analog to Embodiment 48, wherein each linkermoiety independently comprises a peptide from 1 to 75 amino acids inlength, inclusive.

Embodiment 50

The multispecific antibody analog according to any one of Embodiments 48through 49, wherein one or more of the linker moieties independentlycomprises at least one of the 20 naturally occurring amino acids.

Embodiment 51

The multispecific antibody analog according to any one of Embodiments 48through 50, wherein the one or more of the linker moieties independentlycomprises at least one non-natural amino acid incorporated by chemicalsynthesis, post-translational chemical modification or by in vivoincorporation by recombinant expression in a host cell.

Embodiment 52

The multispecific antibody analog according to any one Embodiments of 48through 51, wherein the one or more of the linker moieties independentlycomprises one or more amino acids selected from the group consisting ofserine, glycine, alanine, proline, asparagine, glutamine, glutamate,aspartate, and lysine.

Embodiment 53

The multispecific antibody analog according to any one of Embodiments 48through 52, wherein the one or more of the linker moieties independentlycomprises a majority of amino acids that are sterically unhindered.

Embodiment 54

The multispecific antibody analog according to any one of Embodiments 48through 53, wherein the one or more of the linker moieties independentlycomprises one or more of the following: an acidic linker, a basiclinker, and a structural motif.

Embodiment 55

The multispecific antibody analog according to any one of Embodiments 48through 54, wherein one or more of the linker moieties independentlycomprises: polyglycine, polyalanine, poly(Gly-Ala), or poly(Gly-Ser).

Embodiment 56

The multispecific antibody analog according to any one of Embodiments 48through 55, wherein one or more of the linker moieties independentlycomprises: a polyglycine selected from the group consisting of: (Gly)3(SEQ ID NO: 1), (Gly)4 (SEQ ID NO: 2), and (Gly)5 (SEQ ID NO: 3).

Embodiment 57

The multispecific antibody analog according to any one of Embodiments 48through 56 wherein one or more of the linker moieties independentlycomprises (Gly)₃Lys(Gly)₄ (SEQ ID NO: 4); (Gly)₃AsnGlySer(Gly)₂ (SEQ IDNO: 5); (Gly)₃Cys(Gly)₄ (SEQ ID NO: 6); and GlyProAsnGlyGly (SEQ ID NO:7).

Embodiment 58

The multispecific antibody analog according to any one of Embodiments 48through 57, wherein one or more of the linker moieties independentlycomprises a combination of Gly and Ala.

Embodiment 59

The multispecific antibody analog according to any one of Embodiments 48through 58, wherein one or more of the linker moieties independentlycomprises a combination of Gly and Ser.

Embodiment 60

The multispecific antibody analog according to any one of Embodiments 48through 59, wherein one or more of the linker moieties independentlycomprises a combination of:

Gly and Glu; or

Gly and Asp.

Embodiment 61

The multispecific antibody analog according to any one of 48 through 60,wherein one or more of the linker moieties independently comprises acombination of Gly and Lys.

Embodiment 62

The multispecific antibody analog according to any one of Embodiments 48through 61, wherein one or more of the linker moieties independentlycomprises a sequence selected from group consisting of: [Gly-Ser]_(n)(SEQ ID NO: 8); [Gly-Gly-Ser]_(n) (SEQ ID NO: 9); [Gly-Gly-Gly-Ser]_(n)(SEQ ID NO: 10); [Gly-Gly-Gly-Gly-Ser]_(n) (SEQ ID NO: 11);[Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly]_(n) (SEQ ID NO: 12);[Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly]_(n) (SEQ IDNO: 13); [Gly-Gly-Gly-Gly-SerGly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly]_(n) (SEQ ID NO:14);[Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly]_(n)(SEQ ID NO: 15);[Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly]_(n)(SEQ ID NO: 16);[Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly]_(n)(SEQ ID NO: 17); and combinations thereof; where n is an integerselected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, and 75.

Embodiment 63

The multispecific antibody analog according to any one of Embodiments 48through 62, wherein one or more of the linker moieties independentlycomprises a sequence selected from the group consisting of:[Gly-Glu]_(n) (SEQ ID NO: 18); [Gly-Gly-Glu]_(n) (SEQ ID NO: 19);[Gly-Gly-Gly-Glu]_(n) (SEQ ID NO: 20); [Gly-Gly-Gly-Gly-Glu]_(n) (SEQ IDNO: 21); [Gly-Asp]n (SEQ ID NO: 22); [Gly-Gly-Asp]_(n) (SEQ ID NO: 23);[Gly-Gly-Gly-Asp]_(n) (SEQ ID NO: 24); [Gly-Gly-Gly-Gly-Asp]_(n) (SEQ IDNO: 25); and combinations thereof; where n is an integer selected fromthe group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, and 75.

Embodiment 64

The multispecific antibody analog according to any one of Embodiments 48through 63, wherein at least one of the first and second antigen bindingregions comprises at least one humanized variable heavy domain or atleast one humanized variable light domain.

Embodiment 65

The multispecific antibody analog according to any one of Embodiments 48through 64, wherein at least one of the first and second antigen bindingregions comprises at least one complimentary determining region CDR thatis derived from a non-human multispecific antibody or multispecificantibody fragment.

Embodiment 66

The method according to any one of Embodiments 1 through 45, or themultispecific antibody analog according to any one of Embodiments 46through 65, wherein at least one of the first and second antigen bindingregions binds an epitope from a tumor associated antigen, a hormonereceptor, a cytokine receptor, chemokine receptor, a growth factorreceptor, an immune activating receptor, a hormone, a cytokine, achemokine, a growth factor, a G protein-coupled receptor, or atransmembrane receptor.

Embodiment 67

The method according to any one of Embodiments 1 through 45, or themultispecific antibody analog according to any one of Embodiments 46through 66, wherein at least one of the first and second antigen bindingregions binds a target associated with an autoimmune disorder, aninflammatory disorder, an oncological disorder, neuromuscular disorder,a neurodegenerative disorder, a metabolic disorder, or an infectiousdisease.

Embodiment 68

The method according to any one of Embodiments 1 through 45, or themultispecific antibody analog according to any one of Embodiments 46through 67, wherein the multispecific antibody analog binds at least twodifferent targets.

Embodiment 69

The method according to any one of Embodiments 1 through 45, or themultispecific antibody analog according to any one of Embodiments 46through 68, wherein the multispecific analog binds at least threedifferent targets.

Embodiment 70

The method according to any one of Embodiments 1 through 45, or themultispecific antibody analog according to any one of Embodiments 46through 69, wherein the multispecific antibody analog binds at leastfour different targets.

Embodiment 71

The method according to any one of Embodiments 1 through 45, or themultispecific antibody analog according to any one of Embodiments 46through 70, wherein the multispecific antibody analog binds at least onetarget monovalently.

Embodiment 72

The method according to any one of Embodiments 1 through 45, or themultispecific antibody analog according to any one of Embodiments 46through 71, wherein the multispecific antibody analog binds at least twotargets monovalently.

Embodiment 73

The method according to any one of Embodiments 1 through 45, or themultispecific antibody analog according to any one of Embodiments 46through 72, wherein the multispecific antibody analog binds at leastthree targets monovalently.

Embodiment 74

The method according to any one of Embodiments 1 through 45, or themultispecific antibody analog according to any one of Embodiments 46through 73, wherein the multispecific antibody analog binds at leastfour targets monovalently.

Embodiment 75

The method according to any one of Embodiments 1 through 45, or themultispecific antibody analog according to any one of Embodiments 46through 74, wherein at least one of the antigen binding regionscomprises or is derived from a non-human species.

Embodiment 76

The method according to any one of Embodiments 1 through 45, or themultispecific antibody analog according to any one of Embodiments 46through 75, wherein at least one of the antigen binding sites comprisesa humanized variable domain or a humanized CDR.

Embodiment 77

A method if obtaining or identifying one or more common light chains foruse in preparing a multispecific antibody or multispecific antibodyanalog, the method comprising:

i) performing a first selection against a first antigen from a firstlibrary and obtaining one or more light chains from the output that hasspecificity for the first antigen;

ii) performing a second selection against a second antigen from a secondlibrary and obtaining heavy chains from the output that has specificityfor the second antigen;

iii) generating a restricted library comprising the one or more lightchains obtained in step i) and the heavy chains obtained in step ii);

iv) performing a third selection against the first antigen from therestricted library generated in step iii) and obtaining one or moreantibodies form the output of the third selection, wherein the one ormore antibodies comprise one or more light chains that each havespecificity for the first antigen and the second antigen;

thereby obtaining or identifying the one or more common light chains.

Embodiment 78

The method according to Embodiment 77, wherein:

a) the first library comprises a naïve library;

b) the second library comprises a naïve library; or

c) the first library comprises a naïve library and the second librarycomprises a naïve library.

Embodiment 79

The method according to Embodiment 77 or Embodiment 78, wherein themethod further comprises:

a) performing a subsequent selection against the first antigen from amaturation library;

b) performing a subsequent selection against the second antigen from amaturation library; or

c) performing a subsequent selection against the first antigen from amaturation library and performing a subsequent selection against thesecond antigen from a maturation library;

after performing:

a) step i;

b) step ii

c) step iii; and/or

d) step iv.

Embodiment 80

A method of making a multispecific antibody analog comprising contactingthe one or more common light chains obtained or identified according toany one of Embodiment 77 Embodiment 79 with:

i) a first polypeptide comprising a heavy chain that has specificity forthe first antigen; and

ii) A second polypeptide comprising a heavy chain that has specificityfor the second antigen.

Embodiment 81

The method according to Embodiment 81, wherein the one or more commonlight chains, the first polypeptide, and the second polypeptide areexpressed by host cells.

Embodiment 82

The method according to Embodiment 81 or Embodiment 82, wherein the oneor more common light chains, the first polypeptide, and the secondpolypeptide are expressed by the same host cell.

Embodiment 83

A multispecific antibody analog comprising a common light chain obtainedor identified by performing a method according to any one of Embodiments77 through 81.

Whereas particular embodiments of the invention have been describedabove for purposes of illustration, it will be appreciated by thoseskilled in the art that numerous variations of the details may be madewithout departing from the invention as described in the appendedclaims.

REFERENCES

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What is claimed is:
 1. A method of making a multispecific antibodyanalog comprising at least two first antigen binding regions and atleast two second antigen binding regions, said first and second antigenbinding regions having a common light chain, wherein first antigenbinding regions have a different antigen specificity than the secondantigen binding regions, the method comprising; i) obtaining at leastone light chain from a first antigen binding region having specificityfor the first antigen, wherein the first antigen binding regioncomprises said at least one light chain and a heavy chain; ii) obtainingheavy chains from the output of a selection performed from a naïvelibrary against a second antigen; iii) preparing a restricted librarycomprising heavy chains obtained in step ii) and the at least one lightchain obtained in step i); iv) performing a second selection against thesecond antigen from the library prepared in step iii); v) obtaining anmultispecific antibody comprising the second antigen binding region fromthe selection performed in step iv), wherein the second antigen bindingregion comprises the at least one light chain obtained in step i); vi)incorporating the first antigen binding region and the second antigenbinding region into a multispecific antibody format, wherein the formatcomprises: an IgG moiety comprising either: a) the first antigen bindingregion; or b) the second antigen binding region; and two Fab moieties,wherein each Fab moiety comprises either: a) the second antigen bindingregion; or b) the first antigen binding region; wherein the N-terminusof the heavy chain of one Fab moiety is linked to the C-terminus of theFc region of one heavy chain of the IgG moiety via a linker moiety, andthe N-terminus of the heavy chain of the other Fab moiety is linked tothe C-terminus of the Fc region of the other heavy chain of the IgGmoiety via a linker moiety; thereby generating the multispecificantibody analog.
 2. The method according to claim 1, wherein each linkermoiety independently comprises a peptide from 1 to 75 amino acids inlength, inclusive.
 3. The method according to any one of claims 1 and 2,wherein one or more of the linker moieties independently comprises atleast one of the 20 naturally occurring amino acids.
 4. The methodaccording to any one of claims 1 through 3, wherein the one or more ofthe linker moieties independently comprises at least one non-naturalamino acid incorporated by chemical synthesis, post-translationalchemical modification or by in vivo incorporation by recombinantexpression in a host cell.
 5. The method according to any one claims of1 through 4, wherein the one or more of the linker moietiesindependently comprises one or more amino acids selected from the groupconsisting of serine, glycine, alanine, proline, asparagine, glutamine,glutamate, aspartate, and lysine.
 6. The method according to any one ofclaims 1 through 5, wherein the one or more of the linker moietiesindependently comprises a majority of amino acids that are stericallyunhindered.
 7. The method according to any one of claims 1 through 6,wherein the one or more of the linker moieties independently comprisesone or more of the following: an acidic linker, a basic linker, and astructural motif.
 8. The method according to any one of claims 1 through7, wherein one or more of the linker moieties independently comprises:polyglycine, polyalanine, poly(Gly-Ala), or poly(Gly-Ser).
 9. The methodaccording to any one of claims 1 through 8, wherein one or more of thelinker moieties independently comprises: a polyglycine selected from thegroup consisting of: (Gly)3 (SEQ ID NO: 1), (Gly)4 (SEQ ID NO: 2), and(Gly)5 (SEQ ID NO: 3).
 10. The method according to any one of claims 1through 9 wherein one or more of the linker moieties independentlycomprises (Gly)₃Lys(Gly)₄ (SEQ ID NO: 4); (Gly)₃AsnGlySer(Gly)₂ (SEQ IDNO: 5); (Gly)₃Cys(Gly)₄ (SEQ ID NO: 6); and GlyProAsnGlyGly (SEQ ID NO:7).
 11. The method according to any one of claims 1 through 10, whereinone or more of the linker moieties independently comprises a combinationof Gly and Ala.
 12. The method according to any one of claims 1 through11, wherein one or more of the linker moieties independently comprises acombination of Gly and Ser.
 13. The method according to any one ofclaims 1 through 12, wherein one or more of the linker moietiesindependently comprises a combination of: Gly and Glu; or Gly and Asp.14. The method according to any one of claims 1 through 13, wherein oneor more of the linker moieties independently comprises a combination ofGly and Lys.
 15. The method according to any one of claims 1 through 14,wherein one or more of the linker moieties independently comprises asequence selected from group consisting of: [Gly-Ser]_(n) (SEQ ID NO:8); [Gly-Gly-Ser]_(n) (SEQ ID NO: 9); [Gly-Gly-Gly-Ser]_(n) (SEQ ID NO:10); [Gly-Gly-Gly-Gly-Ser]_(n) (SEQ ID NO: 11);[Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly]_(n) (SEQ ID NO: 12);[Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly]_(n) (SEQ IDNO: 13); [Gly-Gly-Gly-Gly-SerGly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly]_(n) (SEQ ID NO:14);[Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly]_(n)(SEQ ID NO: 15);[Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly]_(n)(SEQ ID NO: 16);[Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly]_(n)(SEQ ID NO: 17); and combinations thereof; where n is an integerselected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, and
 75. 16. The method according toany one of claims 1 through 15, wherein one or more of the linkermoieties independently comprises a sequence selected from the groupconsisting of: [Gly-Glu]_(n) (SEQ ID NO: 18); [Gly-Gly-Glu]_(n) (SEQ IDNO: 19); [Gly-Gly-Gly-Glu]_(n) (SEQ ID NO: 20);[Gly-Gly-Gly-Gly-Glu]_(n) (SEQ ID NO: 21); [Gly-Asp]n (SEQ ID NO: 22);[Gly-Gly-Asp]_(n) (SEQ ID NO: 23); [Gly-Gly-Gly-Asp]_(n) (SEQ ID NO:24); [Gly-Gly-Gly-Gly-Asp]_(n) (SEQ ID NO: 25); and combinationsthereof; where n is an integer selected from the group consisting of 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, and75.
 17. The method according to any one of claims 1 through 16, whereinat least one of the first and second antigen binding regions comprisesat least one humanized variable heavy domain or at least one humanizedvariable light domain.
 18. The method according to any one of claims 1through 17, wherein at least one of the first and second antigen bindingregions comprises at least one complimentary determining region CDR thatis derived from a non-human multispecific antibody or multispecificantibody fragment.
 19. The method according to any one of claims 1through 18, wherein at least one of the first and second antigen bindingregions binds an epitope from a tumor associated antigen, a hormonereceptor, a cytokine receptor, chemokine receptor, a growth factorreceptor, an immune activating receptor, a hormone, a cytokine, achemokine, a growth factor, a G protein-coupled receptor, or atransmembrane receptor.
 20. The method according to any one of claims 1through 19, wherein at least one of the first and second antigen bindingregions binds a target associated with an autoimmune disorder, aninflammatory disorder, an oncological disorder, neuromuscular disorder,a neurodegenerative disorder, a metabolic disorder, or an infectiousdisease.
 21. The method according to any one of claims 1 through 20,wherein the multispecific antibody analog binds at least two differenttargets.
 22. The method according to any one of claims 1 through 21,wherein the multispecific analog binds at least three different targets.23. The method according to any one of claims 1 through 22, wherein themultispecific antibody analog binds at least four different targets. 24.The method according to any one of claims 1 through 23, wherein themultispecific antibody analog binds at least one target monovalently.25. The method according to any one of claims 1 through 24, wherein themultispecific antibody analog binds at least two targets monovalently.26. The multivalent multispecific antibody analog according to any oneof claims 1 through 72, wherein the multispecific antibody analog bindsat least three targets monovalently.
 27. The method according to any oneof claims 1 through 26, wherein the multispecific antibody analog bindsat least four targets monovalently.
 28. The method according to any oneof claims 1 through 27, wherein at least one of the antigen bindingregions comprises or is derived from a non-human species.
 29. The methodaccording to any one of claims 1 through 28, wherein at least one of theantigen binding sites comprises a humanized variable domain or ahumanized CDR.
 30. The method according to any one of claims 1 through29, wherein at least one VH comprises a VH CDR1, a VH CDR2, and a VHCDR3 each independently selected from the following: a VH CDR1 aminoacid sequence selected from the group consisting of: GSVSSGSYYWS;(SEQ ID NO: 26) GSISSGGYYWS; (SEQ ID NO: 27) GSINSSSYYWQ;(SEQ ID NO: 28) FTLSGDWIH; (SEQ ID NO: 29) FNIKDTYIH; (SEQ ID NO: 30)FSLTNYGVH; (SEQ ID NO: 31) GSISSGGDYWQ; (SEQ ID NO: 32)

a VH CDR2 amino acid sequence selected from the group consisting of:YIYYSGSTNYNPSLKS; (SEQ ID NO: 33) IIYYSGWTNYNPSLKS; (SEQ ID NO: 34)EIAYSGSTYYNPSLKS; (SEQ ID NO: 35) EISAAGGYTDYADSVKG; (SEQ ID NO: 36)RIYPTNGYTRYADSVKG; (SEQ ID NO: 37) VIWSGGNTDYNTPFTSR; (SEQ ID NO: 38)

and a VH CDR3 selected from the group consisting of: ARTNLYSTPFDI;(SEQ ID NO: 39) ARGVGPDFWSGYSYSSYFDL; (SEQ ID NO: 40) ARGQQWAAFDI;(SEQ ID NO: 41) ARESRVSFEAAMDY; (SEQ ID NO: 42) SRWGGDGFYAMDY;(SEQ ID NO: 43) RALTYYDYEFAYW. (SEQ ID NO: 44)


31. The method according to any one of claims 1 through 30, wherein atleast one VL comprises a VL CDR1, a VL CDR2, and a VL CDR3 eachindependently selected from the following: a VL CDR1 amino acid sequenceselected from the group consisting of: RASQDISSWLA; (SEQ ID NO: 45)RASQAISSWLA; (SEQ ID NO: 46) RASQNIATDVA; (SEQ ID NO: 47) RASQDVNTAVA;(SEQ ID NO: 48) RASQSIGTNIH;  (SEQ ID NO: 49)

a VL CDR2 amino acid sequence selected from the group consisting of:AASSLQS; (SEQ ID NO: 50) DASSLES; (SEQ ID NO: 51) AASSLQS;(SEQ ID NO: 52) SASFLYS; (SEQ ID NO: 53) YASESIS; (SEQ ID NO: 54)

and a VL CDR3 amino acid sequence selected from the group consisting of:QQEHDFPWT; (SEQ ID NO: 55) HQYQSYSWT; (SEQ ID NO: 56) QQEHDFPWT;(SEQ ID NO: 57) QQSEPEPYT; (SEQ ID NO: 58) QQHYTTPPT; (SEQ ID NO: 59)QQNNNWPTT. (SEQ ID NO: 60)


32. The method according to any one of claims 1 through 31, wherein themultispecific antibody analog comprises at least one heavy chainframework region that corresponds to or is derived from VH1-46, VH3-23,VH4-39, or VH4-61, and wherein at least one light chain framework regionthat corresponds to or is derived from VK1-05, VK1-12, or VK3-11. 33.The method according to any one of claims 1 through 32, wherein themultispecific antibody analog comprises a VH region that comprises anamino acid sequence selected from the group consisting of:(SEQ ID NO: 99) QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCART NLYSTPFDIWGQGTMVTVSS;(SEQ ID NO: 62) QVQLQESGPGLVKPSETLSLTCTVSGGSISSGGYYWSWIRQPPGKGLEWIGIIYYSGWTNYNPSLKSRVTISVDASRNQFSLKLSSVTAADTAVYYCARGVGPDFWSGYSYSSYFDLWGRGTLVTVSS; (SEQ ID NO: 63)QLQLQESGPGLVKPSETLSLTCTVSGGSINSSSYYWQWIRQPPGKGLEWIGEIAYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARG QQWAAFDIWGQGTMVTVSS;(SEQ ID NO: 64) EVQLVESGGGLVQPGGSLRLSCAASGFTLSGDWIHWVRQAPGKGLEWVGEISAAGGYTDYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARESRVSFEAAMDYWGQGTLVTVSS; (SEQ ID NO: 65)EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS;  (SEQ ID NO: 66)QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSDDTAIYYCARALT YYDYEFAYWGQGTLVTVSS;and (SEQ ID NO: 67) QLQLQESGPGLVKPSETLSLTCTVSGGSISSGGDYWQWIRQPPGKGLEWIGEIAYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARG QQWAAFDIWGQGTMVTVSS.


34. The method according to any one of claims 1 through 33, wherein themultispecific antibody analog comprises a VL region that comprises anamino acid sequence selected from the group consisting of:(SEQ ID NO: 68) DIQLTQSPSSVSASVGDRVTITCRASQDISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQEHDFPWTFGG GTKVEIK;(SEQ ID NO: 69) DIQLTQSPSTLSASVGDRVTITCRASQAISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCHQYQSYSWTFGG GTKVEIK;(SEQ ID NO: 70) DIQLTQSPSSVSASVGDRVTITCRASQDISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQEHDFPWTFGG GTKVEIK;(SEQ ID NO: 71) DIQMTQSPSSLSASVGDRVTITCRASQNIATDVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSEPEPYTFGQ GTKVEIK;(SEQ ID NO: 72) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ GTKVEIK; and(SEQ ID NO: 73) DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTHGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGA GTKLELK.


35. The method according to any one of claims 1 through 34, wherein themultispecific antibody analog comprises a polypeptide comprising, fromN-terminus to C-terminus, a first VH region, a CH1, a hinge region, aCH2 region, a CH3 region, a second VH region, and a CH1 region, theamino acid sequence of which comprises an amino acid sequence selectedfrom the group consisting of: (SEQ ID NO: 74)     QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARTNLYSTPFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSQLQLQESGPGLVKPSETLSLTCTVSGGSINSSSYYWQWIRQPPGKGLEWIGEIAYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGQQWAAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC; (SEQ ID NO: 75)     QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARTNLYSTPFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGGSISSGGYYWSWIRQPPGKGLEWIGIIYYSGWTNYNPSLKSRVTISVDASRNQFSLKLSSVTAADTAVYYCARGVGPDFWSGYSYSSYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS C; (SEQ ID NO: 76)     QVQLQESGPGLVKPSETLSLTCTVSGGSISSGGYYWSWIRQPPGKGLEWIGIIYYSGWTNYNPSLKSRVTISVDASRNQFSLKLSSVTAADTAVYYCARGVGPDFWSGYSYSSYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGGSVSSGSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARTNLYSTPFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS C; (SEQ ID NO: 77)     QLQLQESGPGLVKPSETLSLTCTVSGGSINSSSYYWQWIRQPPGKGLEWIGEIAYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGQQWAAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGGSVSSGSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARTNLYSTPFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC.


36. The method according to any one of claims 1 through 35, wherein themultispecific antibody analog comprises four copies of a polypeptidecomprising, from N-terminus to C-terminus, a VL region, and a CK region,and wherein said polypeptide heterodimerizes with compatible VH regionsof the multispecific antibody analog, the amino acid sequence of whichcomprises an amino acid sequence selected from the group consisting of:(SEQ ID NO: 78)      DIQLTQSPSSVSASVGDRVTITCRASQDISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQEHDFPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC;(SEQ ID NO: 79)      DIQLTQSPSTLSASVGDRVTITCRASQAISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCHQYQSYSWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC;(SEQ ID NO: 80)      DIQMTQSPSSLSASVGDRVTITCRASQNIATDVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSEPEPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC;(SEQ ID NO: 81)      DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC;and (SEQ ID NO: 82)      DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTHGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC.


37. The method according to any one of claims 1 through 36 wherein themultispecific antibody analog has binding specificity for an oncologytarget.
 38. The method according to any one of claims 1 through 37,wherein the multispecific antibody analog has binding specificity forone or more targets selected from the group consisting of: EGFR, HER2,and HER3.
 39. The method according to any one of claims 1 through 38,wherein the multispecific antibody analog has binding specificity forEGFR and HER2.
 40. The method according to any one of claims 1 through39, wherein the polypeptide multispecific antibody analog has bindingspecificity for EGFR and HER3.
 41. The method according to any one ofclaims 1 through 40, wherein the multispecific antibody analog hasbinding specificity for EGFR, HER2, and HER3.
 42. The method accordingto any one of claims 1 through 41, wherein the multispecific antibodyanalog is selected from the group consisting of the multispecificantibody analogs described in the Examples.
 43. The method according toany one of claims 1 through 42, wherein the multispecific antibodyanalog is expressed by a prokaryotic host cell or a eukaryotic hostcell.
 44. The method according to any one of claims 1 through 43,wherein the multispecific antibody analog is expressed by a eukaryotichost cell.
 45. The method according to any one of claims 1 through 44,wherein the multispecific antibody analog is expressed by a eukaryotichost cell selected from the group consisting of: yeast cells;Saccharomyces cerevisiae cells; Pichia cells; mammalian cells; Chinesehamster ovary (CHO) cells; human embryonic kidney (HEK) cells; insectcells; Sf9 cells; and Sf21 cells.
 46. A multispecific antibody analogprepared by performing a method according to any one of claims 1 through44.
 47. A multispecific antibody analog comprising at least two firstantigen binding regions and at least two second antigen binding regions,said first and second antigen binding regions having a common lightchain, wherein first antigen binding regions have a different antigenspecificity than the second antigen binding regions.
 48. A multispecificantibody analog comprising at least two first antigen binding regionsand at least two second antigen binding regions, said first and secondantigen binding regions having a common light chain, wherein firstantigen binding regions have a different antigen specificity than thesecond antigen binding regions, wherein the analog prepared is by amethod comprising: i) obtaining at least one light chain from a firstantigen binding region having specificity for the first antigen, whereinthe first antigen binding region comprises said at least one light chainand a heavy chain; ii) obtaining heavy chains from the output of aselection performed from a naïve library against a second antigen; iii)preparing an multispecific antibody library comprising heavy chainsobtained in step ii) and the at least one light chain obtained in stepi); iv) performing a second selection against the second antigen fromthe library prepared in step iii); v) obtaining an multispecificantibody comprising the second antigen binding region from the selectionperformed in step iv); vi) incorporating the first antigen bindingregion and the second antigen binding region into a multispecificantibody format, wherein the format comprises: an IgG moiety comprisingeither: a) the first antigen binding region; or b) the second antigenbinding region; and two Fab moieties, wherein each Fab moiety compriseseither: a) the second antigen binding region; or b) the first antigenbinding region; wherein the N-terminus of the heavy chain of one Fabmoiety is linked to the C-terminus of the Fc region of one heavy chainof the IgG moiety via a linker moiety, and the N-terminus of the heavychain of the other Fab moiety is linked to the C-terminus of the Fcregion of the other heavy chain of the IgG moiety via a linker moiety;thereby generating the multispecific antibody analog.
 49. Themultispecific antibody analog to claim 48, wherein each linker moietyindependently comprises a peptide from 1 to 75 amino acids in length,inclusive.
 50. The multispecific antibody analog according to any one ofclaims 48 through 49, wherein one or more of the linker moietiesindependently comprises at least one of the 20 naturally occurring aminoacids.
 51. The multispecific antibody analog according to any one ofclaims 48 through 50, wherein the one or more of the linker moietiesindependently comprises at least one non-natural amino acid incorporatedby chemical synthesis, post-translational chemical modification or by invivo incorporation by recombinant expression in a host cell.
 52. Themultispecific antibody analog according to any one claims of 48 through51, wherein the one or more of the linker moieties independentlycomprises one or more amino acids selected from the group consisting ofserine, glycine, alanine, proline, asparagine, glutamine, glutamate,aspartate, and lysine.
 53. The multispecific antibody analog accordingto any one of claims 48 through 52, wherein the one or more of thelinker moieties independently comprises a majority of amino acids thatare sterically unhindered.
 54. The multispecific antibody analogaccording to any one of claims 48 through 53, wherein the one or more ofthe linker moieties independently comprises one or more of thefollowing: an acidic linker, a basic linker, and a structural motif. 55.The multispecific antibody analog according to any one of claims 48through 54, wherein one or more of the linker moieties independentlycomprises: polyglycine, polyalanine, poly(Gly-Ala), or poly(Gly-Ser).56. The multispecific antibody analog according to any one of claims 48through 55, wherein one or more of the linker moieties independentlycomprises: a polyglycine selected from the group consisting of: (Gly)3(SEQ ID NO: 1), (Gly)4 (SEQ ID NO: 2), and (Gly)5 (SEQ ID NO: 3). 57.The multispecific antibody analog according to any one of claims 48through 56 wherein one or more of the linker moieties independentlycomprises (Gly)₃Lys(Gly)₄ (SEQ ID NO: 4); (Gly)₃AsnGlySer(Gly)₂ (SEQ IDNO: 5); (Gly)₃Cys(Gly)₄ (SEQ ID NO: 6); and GlyProAsnGlyGly (SEQ ID NO:7).
 58. The multispecific antibody analog according to any one of claims48 through 57, wherein one or more of the linker moieties independentlycomprises a combination of Gly and Ala.
 59. The multispecific antibodyanalog according to any one of claims 48 through 58, wherein one or moreof the linker moieties independently comprises a combination of Gly andSer.
 60. The multispecific antibody analog according to any one ofclaims 48 through 59, wherein one or more of the linker moietiesindependently comprises a combination of: Gly and Glu; or Gly and Asp.61. The multispecific antibody analog according to any one of 48 through60, wherein one or more of the linker moieties independently comprises acombination of Gly and Lys.
 62. The multispecific antibody analogaccording to any one of claims 48 through 61, wherein one or more of thelinker moieties independently comprises a sequence selected from groupconsisting of: [Gly-Ser]_(n) (SEQ ID NO: 8); [Gly-Gly-Ser]_(n) (SEQ IDNO: 9); [Gly-Gly-Gly-Ser]_(n) (SEQ ID NO: 10); [Gly-Gly-Gly-Gly-Ser]_(n)(SEQ ID NO: 11); [Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly]_(n) (SEQ ID NO:12); [Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly]_(n) (SEQID NO: 13); [Gly-Gly-Gly-Gly-SerGly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly]_(n) (SEQ ID NO:14);[Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly]_(n)(SEQ ID NO: 15);[Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly]_(n)(SEQ ID NO: 16);[Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly]_(n)(SEQ ID NO: 17); and combinations thereof; where n is an integerselected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, and
 75. 63. The multispecificantibody analog according to any one of claims 48 through 62, whereinone or more of the linker moieties independently comprises a sequenceselected from the group consisting of: [Gly-Glu]_(n) (SEQ ID NO: 18);[Gly-Gly-Glu]_(n) (SEQ ID NO: 19); [Gly-Gly-Gly-Glu]_(n) (SEQ ID NO:20); [Gly-Gly-Gly-Gly-Glu]_(n) (SEQ ID NO: 21); [Gly-Asp]n (SEQ ID NO:22); [Gly-Gly-Asp]_(n) (SEQ ID NO: 23); [Gly-Gly-Gly-Asp]_(n) (SEQ IDNO: 24); [Gly-Gly-Gly-Gly-Asp]_(n) (SEQ ID NO: 25); and combinationsthereof; where n is an integer selected from the group consisting of 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, and75.
 64. The multispecific antibody analog according to any one of claims48 through 63, wherein at least one of the first and second antigenbinding regions comprises at least one humanized variable heavy domainor at least one humanized variable light domain.
 65. The multispecificantibody analog according to any one of claims 48 through 64, wherein atleast one of the first and second antigen binding regions comprises atleast one complimentary determining region CDR that is derived from anon-human multispecific antibody or multispecific antibody fragment. 66.The method according to any one of claims 1 through 45, or themultispecific antibody analog according to any one of claims 46 through65, wherein at least one of the first and second antigen binding regionsbinds an epitope from a tumor associated antigen, a hormone receptor, acytokine receptor, chemokine receptor, a growth factor receptor, animmune activating receptor, a hormone, a cytokine, a chemokine, a growthfactor, a G protein-coupled receptor, or a transmembrane receptor. 67.The method according to any one of claims 1 through 45, or themultispecific antibody analog according to any one of claims 46 through66, wherein at least one of the first and second antigen binding regionsbinds a target associated with an autoimmune disorder, an inflammatorydisorder, an oncological disorder, neuromuscular disorder, aneurodegenerative disorder, a metabolic disorder, or an infectiousdisease.
 68. The method according to any one of claims 1 through 45, orthe multispecific antibody analog according to any one of claims 46through 67, wherein the multispecific antibody analog binds at least twodifferent targets.
 69. The method according to any one of claims 1through 45, or the multispecific antibody analog according to any one ofclaims 46 through 68, wherein the multispecific analog binds at leastthree different targets.
 70. The method according to any one of claims 1through 45, or the multispecific antibody analog according to any one ofclaims 46 through 69, wherein the multispecific antibody analog binds atleast four different targets.
 71. The method according to any one ofclaims 1 through 45, or the multispecific antibody analog according toany one of claims 46 through 70, wherein the multispecific antibodyanalog binds at least one target monovalently.
 72. The method accordingto any one of claims 1 through 45, or the multispecific antibody analogaccording to any one of claims 46 through 71, wherein the multispecificantibody analog binds at least two targets monovalently.
 73. The methodaccording to any one of claims 1 through 45, or the multispecificantibody analog according to any one of claims 46 through 72, whereinthe multispecific antibody analog binds at least three targetsmonovalently.
 74. The method according to any one of claims 1 through45, or the multispecific antibody analog according to any one of claims46 through 73, wherein the multispecific antibody analog binds at leastfour targets monovalently.
 75. The method according to any one of claims1 through 45, or the multispecific antibody analog according to any oneof claims 46 through 74, wherein at least one of the antigen bindingregions comprises or is derived from a non-human species.
 76. The methodaccording to any one of claims 1 through 45, or the multispecificantibody analog according to any one of claims 46 through 75, wherein atleast one of the antigen binding sites comprises a humanized variabledomain or a humanized CDR.
 77. A method if obtaining or identifying oneor more common light chains for use in preparing a multispecificantibody or multispecific antibody analog, the method comprising: i)performing a first selection against a first antigen from a firstlibrary and obtaining one or more light chains from the output that hasspecificity for the first antigen; ii) performing a second selectionagainst a second antigen from a second library and obtaining heavychains from the output that has specificity for the second antigen; iii)generating a restricted library comprising the one or more light chainsobtained in step i) and the heavy chains obtained in step ii); iv)performing a third selection against the first antigen from therestricted library generated in step iii) and obtaining one or moreantibodies form the output of the third selection, wherein the one ormore antibodies comprise one or more light chains that each havespecificity for the first antigen and the second antigen; therebyobtaining or identifying the one or more common light chains.
 78. Themethod according to claim 77, wherein: a) the first library comprises anaïve library; b) the second library comprises a naïve library; or c)the first library comprises a naïve library and the second librarycomprises a naïve library.
 79. The method according to claim 77 or claim78, wherein the method further comprises: a) performing a subsequentselection against the first antigen from a maturation library; b)performing a subsequent selection against the second antigen from amaturation library; or c) performing a subsequent selection against thefirst antigen from a maturation library and performing a subsequentselection against the second antigen from a maturation library; afterperforming: a) step i; b) step ii c) step iii; and/or d) step iv.
 80. Amethod of making a multispecific antibody analog comprising contactingthe one or more common light chains obtained or identified according toany one of claims 77 through 79 with: i) a first polypeptide comprisinga heavy chain that has specificity for the first antigen; and ii) Asecond polypeptide comprising a heavy chain that has specificity for thesecond antigen.
 81. The method according to claim 81, wherein the one ormore common light chains, the first polypeptide, and the secondpolypeptide are expressed by host cells.
 82. The method according toclaim 81 or claim 82, wherein the one or more common light chains, thefirst polypeptide, and the second polypeptide are expressed by the samehost cell.
 83. A multispecific antibody analog comprising a common lightchain obtained or identified by performing a method according to any oneof claims 77 through 81.